Bacillus thuringiensis RTI545 compositions and methods of use for benefiting plant growth and controlling plant pests

ABSTRACT

Compositions are provided that include a new Bacillus thuringiensis strain designated RTI545 for use in benefiting plant growth and controlling plant pests. In particular, the RTI545 strain is useful for controlling plant nematode, insect and fungal pests. The compositions include plant seeds coated with the RTI545 strain. The compositions can be applied alone or in combination with other microbial, biological, or chemical insecticides, fungicides, nematicides, bacteriocides, herbicides, plant extracts, plant growth regulators, or fertilizers. In one example, enhanced growth and insect control are provided by delivering at the time of planting a combination of a chemical insecticide such as bifenthrin and a liquid fertilizer to plants or seeds treated with RTI545.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional application No.62/404,275, filed Oct. 5, 2016, the disclosure of which is herebyincorporated herein by reference in its entirety.

TECHNICAL FIELD

The presently disclosed subject matter relates to compositionscomprising an isolated Bacillus thuringiensis bacterial strain forapplication to plant seeds and roots, and the soil surrounding plants tobenefit plant growth and to control plant pests.

BACKGROUND

A number of microorganisms having beneficial effects on plant growth andhealth are known to be present in the soil, to live in association withplants specifically in the root zone (rhizosphere-associated bacteria),or to reside as endophytes within the plant. Their beneficial plantgrowth promoting properties include nitrogen fixation, iron chelation,phosphate solubilization, inhibition of non-beneficial microorganisms,resistance to, or exclusion of pests, Induced Systemic Resistance (ISR),Systemic Acquired Resistance (SAR), decomposition of plant material insoil to increase useful soil organic matter, and synthesis ofphytohormones such as indole-acetic acid (IAA), acetoin and2,3-butanediol that stimulate plant growth, development and responses toenvironmental stresses such as drought. In addition, thesemicroorganisms can interfere with a plant's ethylene stress response bybreaking down the precursor molecule, 1-aminocyclopropane-1-carboxylate(ACC), thereby stimulating plant growth and slowing fruit ripening.These beneficial microorganisms can improve soil quality, plant growth,yield, and quality of crops. Various microorganisms exhibit biologicalactivity such as to be useful to control plant diseases. Suchbiopesticides (living organisms and the compounds naturally produced bythese organisms) are generally considered safer and more biodegradablethan synthetic fertilizers and pesticides.

For example, beneficial plant associated bacteria, both rhizospheric andendophytic, are known to provide a multitude of benefits to host plantsthat ranges from resistance to diseases and insects pests and toleranceto environmental stresses including cold, salinity and drought stress.As the plants with inoculated plant growth promoting bacteria acquiremore water and nutrients from soils, e.g. due to a better developed rootsystem, the plants grow healthier and are less susceptible to biotic andabiotic stresses. As such the microbial compositions of the presentinvention can be applied alone or in combination with current cropmanagement inputs such as chemical fertilizers, herbicides, andpesticides to maximize crop productivity. Plant growth promoting effectstranslate into faster growing plants and increase above ground biomass,a property that can be applied to improve early vigor. One benefit ofimproved early vigor is that plants are more competitive and out-competeweeds, which directly reduces the cost for weed management by minimizinglabor and herbicide application. Plant growth promoting effects alsotranslate into improved root development, including deeper and widerroots with more fine roots that are involved in the uptake of water andnutrients. This property allows for better use of agriculturalresources, and a reduction in water used in irrigation needs and/orfertilizer application. Changes in root development and rootarchitecture affect the interactions of the plant with other soil-bornemicroorganisms, including beneficial fungi and bacteria that help theplant with nutrient uptake including nitrogen fixation and phosphatesolubilization. These beneficial microbes also compete against plantpathogens to increase overall plant health and decrease the need forchemical fungicides and pesticides. A more developed root system alsoallows for improved yields when pests are present.

Fungal phytopathogens, including but not limited to Botrytis spp. (e.g.Botrytis cinerea), Fusarium spp. (e.g. F. oxysporum and F. graminearum),Rhizoctonia spp. (e.g. R. solani), Magnaporthe spp., Mycosphaerellaspp., Puccinia spp. (e.g. P. recondita), Phytopthora spp. and Phakopsoraspp. (e.g. P. pachyrhizi), are one type of plant pest that can causesevere economic losses in the agricultural and horticultural industries.Chemical agents can be used to control fungal phytopathogens, but theuse of chemical agents suffers from disadvantages including high cost,emergence of resistant strains of pests, and potentially undesirableenvironmental impacts. In addition, such chemical treatments mayadversely affect beneficial bacteria, fungi, and arthropods in additionto the plant pest at which the treatments are targeted. A second type ofplant pest are bacterial pathogens, including but not limited to Erwiniaspp. (such as Erwinia chrysanthemi), Pantoea spp. (such as P. citrea),Xanthomonas (e.g. Xanthomonas campestris), Pseudomonas spp. (such as P.syringae) and Ralstonia spp. (such as R. soleacearum) that cause severeeconomic losses in the agricultural and horticultural industries.Similar to pathogenic fungi, the use of chemical agents to treat thesebacterial pathogens suffers from disadvantages. Viruses and virus-likeorganisms comprise a third type of plant disease-causing agent that ishard to control, but to which bacterial microorganisms can provideresistance in plants via induced systemic resistance (ISR). Thus,microorganisms that can be applied as biofertilizer and/or biopesticideto control pathogenic fungi, viruses, and bacteria are desirable and inhigh demand to improve agricultural sustainability. A final type ofplant pathogen includes plant pathogenic nematodes and insects, whichcan cause severe damage and loss of plants and reductions in yield.

Some members of the species Bacillus have been reported as biocontrolstrains, and some have been applied in commercial products (Joseph W.Kloepper, et al. 2004, Phytopathology Vol. 94, No. 11, 1259-1266). Forexample, strains currently being used in commercial biocontrol productsinclude: Bacillus pumilus strain QST2808, used as active ingredient inSONATA and BALLAD-PLUS, produced by BAYER CROP SCIENCE; Bacillus pumilusstrain GB34, used as active ingredient in YIELDSHIELD, produced by BAYERCROP SCIENCE; Bacillus subtilis strain QST713, used as the activeingredient of SERENADE, produced by BAYER CROP SCIENCE; Bacillussubtilis strain GBO3, used as the active ingredient in KODIAK andSYSTEM3, produced by HELENA CHEMICAL COMPANY. Various strains ofBacillus thuringiensis and Bacillus firmus have been applied asbiocontrol agents against nematodes and insects and these strains serveas the basis of numerous commercially available biocontrol products,including DIPEL comprising a Bacillus thuringiensis subsp. kurstakistrain, produced by VALENT BIOSCIENCES CORPORATION, and NORTICA andPONCHO-VOTIVO comprising a B. firmus strain, produced by BAYER CROPSCIENCE. In addition, Bacillus strains currently being used incommercial biostimulant products include: Bacillus subtilis var.amyloliquefaciens strain FZB42 used as the active ingredient inRHIZOVITAL 42, produced by ABiTEP GmbH, as well as various otherBacillus subtilis species that are included as whole cells includingtheir fermentation extract in biostimulant products, such as FULZYMEproduced by JHBiotech Inc.

However, it is desirable to develop new compositions and methods forbenefiting plant growth and controlling plant pests.

SUMMARY

The presently disclosed subject matter provides microbial compositionsand methods for their use in benefiting plant growth and controllingplant pests.

In one embodiment, a composition is provided comprising a biologicallypure culture of Bacillus thuringiensis RTI545 deposited as ATCC No.PTA-122161, or a mutant thereof having all the identifyingcharacteristics thereof, for application to a plant for one or both ofbenefiting plant growth or conferring protection against a plant pest ina susceptible plant.

In one embodiment, a method is provided for one or both of benefitinggrowth of a plant or conferring protection against a plant pest in asusceptible plant, the method comprising delivering a compositioncomprising a biologically pure culture of Bacillus thuringiensis RTI545deposited as ATCC No. PTA-122161, or a mutant thereof having all theidentifying characteristics thereof to a plant, plant part, seed of theplant, roots of the plant, soil or growth medium surrounding the plantor the seed of the plant, or soil or growth medium before planting theplant or sowing seed of the plant, in an amount suitable to benefit theplant growth and/or to confer protection against the plant pest in thesusceptible plant.

In one embodiment, a method is provided for one or both of benefitinggrowth of a plant or conferring protection against a plant pest in asusceptible plant, the method comprising: delivering to the plant, plantpart, seed of the plant, roots of the plant, soil or growth mediumsurrounding the plant or the seed of the plant, or soil or growth mediumbefore planting the plant or sowing seed of the plant, a combination of:a composition comprising a biologically pure culture of Bacillusthuringiensis RTI545 deposited as ATCC No. PTA-122161, or a mutantthereof having all the identifying characteristics thereof in an amountsuitable to benefit the plant growth and/or to confer protection againstthe plant pest in the susceptible plant; and one or a combination of aninsecticide, a fungicide, nematicide, bacteriocide, biostimulant,herbicide, plant extract, microbial extract, plant growth regulator, orfertilizer, in an amount suitable to benefit the plant growth and/or toconfer protection against the plant pest in the susceptible plant. Eachof these additional agents can be either a biological agent or achemical agent.

In one embodiment, a plant seed is provided coated with a compositioncomprising spores of a biologically pure culture of Bacillusthuringiensis RTI545 deposited as ATCC No. PTA-122161, or a mutantthereof having all the identifying characteristics thereof, present inan amount suitable to benefit plant growth and/or to confer protectionagainst a plant pest in a susceptible plant.

In one embodiment, a method is provided for one or both of benefitinggrowth of a plant or conferring protection against a plant pest in asusceptible plant, the method comprising planting a seed of the plant,wherein the seed has been coated with a composition comprising abiologically pure culture of Bacillus thuringiensis RTI545 deposited asATCC PTA-122161, or a mutant thereof having all the identifyingcharacteristics thereof, wherein growth of the plant from the seed isbenefited and/or protection against the plant pest is conferred.

In one embodiment, a composition is provided for benefiting plantgrowth, the composition comprising: a biologically pure culture ofBacillus thuringiensis s RTI545 deposited as ATCC No. PTA-122161, or amutant thereof having all the identifying characteristics thereof; and abifenthrin insecticide.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying Figures describedbelow.

FIG. 1A is a schematic diagram showing on the far left a plant seed(inner circle) coated with a chemical insecticide (dark band surroundinginner circle) with plant insect pests in the plant rhizosphererepresented by horizontal marks. The middle portion of the diagram showsthe sprouted plant seed with diffused insecticide protecting the rootsof the plant seed from the insect pests (protection represented by the“X” marks). The far right of the diagram shows diminished protection ofthe roots of the plant seed from the insect pests as the roots growbeyond the diffusion zone of the chemical insecticide.

FIG. 1B shows the schematic diagram of FIG. 1A with the addition ofBacillus thuringiensis RTI545 to the coating on the plant seed or to thesoil surrounding the plant seed according to one or more embodiments ofthe present disclosure. The far right of the diagram shows continuedprotection of the roots of the plant seed from the insect pests even asthe roots grow beyond the diffusion zone of the chemical insecticide asa result of the establishment of Bacillus thuringiensis RTI545 in theplant rhizosphere.

FIG. 2 is a schematic drawing showing the phylogeny of the RTI545 strainusing the housekeeping gene rpoB according to one or more embodiments ofthe present disclosure.

Bootstrap values indicate replicates of 1000. Outgroup sequences arefrom the hypertheromiphilic archaea Pyrococcus furiosus DSM3638.

FIG. 3A is an image of corn seedlings taken 12 days from planting ofseed treated with vegetative cells of Bacillus thuringiensis strainRTI545 at planting by drench irrigation according to one or moreembodiments of the present disclosure.

FIG. 3B is an image of corn seedlings taken 12 days from planting ofseed treated similarly to that in FIG. 3A but without the addition ofthe Bacillus thuringiensis RTI545 cells.

FIG. 4 is an image showing the ability of Bacillus thuringiensis RTI545cells to repel Southern corn rootworm (SCRW) larvae in a choice feedingassay of corn seedlings according to one or more embodiments of thepresent disclosure. In the image, the filter paper on the left wastreated with Bacillus thuringiensis strain RTI545 cells and the filterpaper on the right was treated with water as a control.

FIG. 5 is a graph showing the number of cysts per gram of root biomassof potato plants potted in soil naturally infected with Globodera sp.nematodes and enhanced with 10⁹ cfu spores per liter soil Bacillusthuringiensis RTI545 cells (RTI545) as compared to control and soiltreatments: Vydate (DUPONT; A.I.=Oxamyl [MethoylN′N′-dimethyl-N-[(methyl carbamoyl)oxyl)oxy]-1-thiooxamimidate), BIOACT(BAYER CROPSCIENCES LP; Paecilomyces lilacinus strain 251), CAREX(NUFARM, pyridaben), and HD-1 (Bacillus thuringiensis subsp. kurstakiHD-1) according to one or more embodiments of the present disclosure.

FIG. 6A shows a schematic plan drawing of a chemotaxis test arena forassaying attraction/repellency of test samples to nematodes. FIG. 6B isa photograph of an assay of kanosamine tested at 100 μg/ml, wherein dotsrepresenting nematode locations indicate neutral distribution. FIG. 6Cis a photograph of an assay of RTI545 supernatent tested at 100%strength, wherein dots representing nematode locations indicaterepellant distribution.

DETAILED DESCRIPTION

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, the term“about” when used in connection with one or more numbers or numericalranges, should be understood to refer to all such numbers, including allnumbers in a range and modifies that range by extending the boundariesabove and below the numerical values set forth. The recitation ofnumerical ranges by endpoints includes all numbers, e.g., wholeintegers, including fractions thereof, subsumed within that range (forexample, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well asfractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and anyrange within that range. When a range is recited as being from a list oflower limits to a list of upper limits, ranges are defined as being fromany one of the recited lower limits to any one of the recited upperlimits.

Tradenames are indicated herein in UPPERCASE.

As used herein for the purposes of this specification and claims, in oneembodiment, the phrase “a biologically pure culture of Bacillusthuringiensis RTI545” refers to one or a combination of: spores of abiologically pure fermentation culture of the bacterial strain,vegetative cells of a biologically pure fermentation culture of thebacterial strain, one or more products of a biologically purefermentation culture of the bacterial strain, a culture solid of abiologically pure fermentation culture of the bacterial strain, aculture supernatant of a biologically pure fermentation culture of thebacterial strain, and a cell-free extract of a biologically purefermentation culture of the bacterial strain.

In another embodiment, the phrase “a biologically pure culture ofBacillus thuringiensis RTI545” refers to one or a combination of: sporesof a biologically pure fermentation culture of the bacterial strain,vegetative cells of a biologically pure fermentation culture of thebacterial strain, one or more products of a biologically purefermentation culture of the bacterial strain, and a culture solid of abiologically pure fermentation culture of the bacterial strain. In onevariant of this embodiment, the phrase may refer to the spores of abiologically pure fermentation culture of the bacterial strain.

In still another embodiment, the phrase “a biologically pure culture ofBacillus thuringiensis RTI545” refers to one or a combination of: aculture supernatant of a biologically pure fermentation culture of thebacterial strain, and a cell-free extract of a biologically purefermentation culture of the bacterial strain.

Notably, the “a biologically pure culture of Bacillus thuringiensisRTI545” may be in the form of spores, vegetative cells or cell-freeextracts of the biologically pure culture.

As used herein for the purposes of this specification and claims, thephrase “a plant pest” refers to any pest that is harmful and/orpathogenic to a plant, including without limitation, a plant pest suchas an insect, a parasite, a nematode, a fungus, a bacteria, or a virus.

A new plant-associated bacterium isolated from the soil of fescue grassis provided herein and referred to as “RTI545”. The strain wasidentified as being a Bacillus thuringiensis strain based on sequenceanalysis, although the strain lacks the genes for crystal proteins oftenfound in B. thuringiensis strains. Unexpectedly, the RTI545 straindemonstrates strong insect repelling activity, but fails to kill theinsect larvae in direct contact and choice feeding assays. Compositionsand methods are provided herein that include a biologically pure cultureof the Bacillus thuringiensis RTI545 strain for delivery to a plant,plant part, plant seed, plant roots, or soil to benefit plant growth andconfer protection against plant pests. The growth benefits and conferredprotection include improved seedling vigor, improved root development,improved plant growth, improved plant health, increased yield, improvedappearance, improved resistance to plant pests, reduced pathogenicinfection, or a combination thereof.

Delivery of the composition to the plant or plant part includes deliveryto any portion of the plant, including above-ground portions, such asfoliar parts, and parts of plants for propagating the plant such asseedlings, transplants, cuttings (e.g. stems, roots, leaves, and thelike), rhizomes, spores, setts (e.g. of sugarcane), bulbs, corms,tubers, or portions thereof, or other plant tissue from which a completeplant can be obtained. Delivery to the soil or growth medium surroundingthe plant or the seed of the plant, or soil or growth medium beforeplanting the plant or sowing seed of the plant includes in-furrowapplications of the composition at the time of planting, includesincorporation or mixing of the composition with the soil or growthmedium, application of the composition to the surface of the soil orgrowth medium such as by soil drench, and the like.

In one embodiment, the Bacillus thuringiensis RTI545 strain is deliveredto a plant, plant part, plant seed, plant roots or soil in combinationwith a chemical insecticide to extend the control of the chemicalinsecticide through establishment of the RTI545 strain in the plantrhizosphere. A proposed mechanism of this insect control by the RTI545strain is illustrated in FIG. 1. FIG. 1A shows insect control by coatinga seed with a chemical insecticide alone (i.e., without RTI545). A plantseed (inner circle) coated with an insecticide (dark band surroundinginner circle) is shown on the far left of the FIG. 1A diagram, which issurrounded by plant insect pests in the plant rhizosphere represented byhorizontal marks. The middle portion of the diagram shows the sproutedplant seed with diffused insecticide protecting the roots of the plantseed from the insect pests (protection represented by the “X” marks).The far right of the diagram shows diminished protection of the roots ofthe plant seed from the insect pests as the roots grow beyond thediffusion zone of the chemical insecticide. The diagram in FIG. 1Billustrates a proposed mechanism for how addition of Bacillusthuringiensis RTI545 spores to the coating on the plant seed or as anin-furrow application improves insect control over use of theinsecticide coating alone. Specifically, the far right side of the FIG.1B diagram shows continued protection of the roots of the plant seedfrom the insect pests even as the roots grow beyond the diffusion zoneof the chemical insecticide as a result of the establishment of Bacillusthuringiensis RTI545 in the plant rhizosphere. In one example, seedscoated with RTI545 cells or otherwise treated with RTI545 are planted incombination with the chemical insecticide, bifenthrin, and applicationof a liquid fertilizer to benefit plant growth and control insect pests.

The isolation and characterization of the RTI545 strain is describedmore specifically in the EXAMPLEs provided herein. EXAMPLE 1 describescomparison of the sequences of the 16S rDNA (SEQ ID NO.: 1) and rpoB(SEQ ID NO.: 2) genes of the RTI545 strain to those of other knownbacterial strains in the NCBI and RDP databases using BLAST. Thisanalysis placed strain RTI545 within the Bacilluscereus/thuringiensis/anthracis clade. Further phylogenetic analysis ofthe RTI545 strain and relevant Bacillus species was performed usingBootstrap consensus trees (1000 replicates) on the rpoB gene. Theconsensus tree for the rpoB gene is shown in FIG. 2. As can be seen inFIG. 2, the RTI545 strain forms a separate branch in the Bacilluscereus/thuringiensis/anthracis clade indicating that RTI545 is a newstrain falling within the Bacillus cereus/thuringiensis/anthracis clade.Additional sequence analysis revealed that the RTI545 strain lacks thegenes for crystal proteins often found in B. thuringiensis strains.

In addition, whole genome sequence analysis was performed to compare theRTI545 strain with closely related strains of the Bacillus species usingboth MUMmer- and BLASTn-based Average Nucleotide Identity (ANI) andUNIPEPT analysis to confirm its phylogenetic classification. The resultsof the MUMmer and BLASTn based ANI calculations are shown in Table Ibelow. Both the ANI and UNIPEPT (data not shown) analyses revealed asignificant degree of sequence similarity between RTI545 and publishedsequences of strains indicated as both B. cereus and B. thuringiensis.The highest sequence similarity to a recognized type strain is to therecognized type strain B. thuringiensis Berliner ATCC10792. Again thedifferences in whole genome sequence from those previously publishedindicate that RTI545 is a new Bacillus thuringiensis strain fallingwithin the Bacillus cereus/thuringiensis/anthracis clade.

Based on the foregoing sequence analyses, the RTI545 strain wasidentified as a new strain, and is herein referred to as a Bacillusthuringiensis strain.

The strain of RTI545 was deposited on May 12, 2015 under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure at the American TypeCulture Collection (ATCC) in Manassas, Va., USA and bears the PatentAccession No. PTA-122161. All restrictions upon availability to thepublic will be irrevocably removed upon granting of the patent.

Experimental results demonstrating the growth promoting, antimicrobial,and insect and nematode and fungal control activities of the Bacillusthuringiensis RTI545 strain in various plants and under varyingconditions including in vitro, greenhouse and field trial studies areprovided in FIGS. 3-5 and in EXAMPLES 2-17 herein. Specifically, theBacillus thuringiensis RTI545 strain is shown to benefit plant growthand confer control against plant pests including rootworms such asSouthern corn rootworm (SCRW), wireworms such as wheat wireworms andcorn wireworms, white grub complex pests, soil-dwelling maggots such asseed corn maggots and seed maggots, plant bugs such as Western plant bug(WPB), nematodes and fungal pathogens such as Rhizoctonia spp. In somecases, seed treatment with the Bacillus thuringiensis RTI545 strainprovided equivalent or superior results as compared to commerciallyavailable products based on a combination of biological and chemicalactive agents or chemical active agents alone.

The antagonistic properties of the Bacillus thuringiensis RTI545 againstseveral major plant pathogens in plate assays are described in EXAMPLE 2and phenotypic traits such as phytohormone production, acetoin andindole acetic acid (IAA), and nutrient cycling of the strain aredescribed in EXAMPLE 3.

EXAMPLE 4 describes the positive effects of incubation of corn seed withRTI545 cells on seed germination, root development, and early growth.The results are shown in FIG. 3A and FIG. 3B, which are images of thecorn seedlings after 12 days grown in the presence (FIG. 3A) and absence(FIG. 3B) of the RTI545 strain. As can be seen in the figures, thepresence of the RTI545 strain resulted in a significant growthadvantage.

EXAMPLE 5 describes the positive effects of inoculation of corn seedwith RTI545 cells on early plant growth and vigor. Surface sterilizedgerminated corn seeds were inoculated for 2 days in a suspension of 10⁸CFU/ml of RTI545 at room temperature and, subsequently, the inoculatedseeds were planted in pots and incubated in a greenhouse. The wet anddry weight of the corn shoot biomass was measured after 42 days growth.Wet weight and dry weight of the corn shoot biomass increased for theplants inoculated with the Bacillus thuringiensis RTI545 strain comparedto the non-inoculated control. As can be discerned from the significantincrease in both wet and dry biomass, the presence of the RTI545 strainresulted in a significant growth advantage.

EXAMPLE 6 describes the unexpected insect repelling activity of theRTI545 strain. For antagonism against Western plant bug (WPB), Bacillusthuringiensis RTI545 was evaluated in direct spray, choice feeding, andno-choice feeding assays along with controls including a media blank,chemical active agent (O,S-Dimethyl acetylphosphoramidothioate), andBacillus thuringiensis subsp. kurstaki HD-1 (HD-1). As expected, nosignificant mortality (direct spray and no-choice feeding assays) orrepellency (choice feeding assay) was observed for the medium blank orHD-1 treatments, while the chemical control killed (direct spray andno-choice feeding assays) and repelled (choice feeding assay) the WPB.Bacillus thuringiensis RTI545 provided no significant mortality to WPBwhen applied in both direct spray and in no-choice feeding assays;however, unexpectedly, RTI545 displayed a repellent behavior at 124hours after WPB were placed into choice assay arenas. Specifically, whenthe WPB were placed into a container containing a treated and anon-treated food source, WPB were observed to be feeding only on thenon-treated food source (data not shown).

For antagonism against Southern corn rootworm (SCRW) larvae, Bacillusthuringiensis RTI545 cells were evaluated in a choice feeding assay ofcorn seedlings and compared to a water control. Additional treatmentscompared to the water control were i) strain Bacillus thuringiensissubsp. kurstaki HD-1 (HD-1), ii) chemical control CAPTURE LFR(A.I.=17.15% bifenthrin), and iii) 869 medium. Filter paper was cut inhalf and each section placed in a petri dish, to which either treatmentor deionized water was applied to each half of the paper. One germinatedcorn seed was situated on each moist filter paper half. Tensecond-instar larvae were placed at the midline between treated anduntreated filter paper. Dishes were sealed and maintained for 6 days. Animage of the plate assay with the RTI545 cells after 6 days is shown inFIG. 4, and the data from all of the plate assays are summarized inTable IV. As was observed in the assay above for WPB, the RTI545unexpectedly repelled, but did not kill the SCRW larvae. As can be seenin FIG. 4 and Table IV, the RTI545 cultures were excellent at repellingthe SCRW larvae; 100% of the larvae were present on the water-treatedhalf of the filter paper and none of the larvae on the RTI545 treatedpaper. In contrast, the larvae were statistically evenly divided betweentreatment and water control for the HD-1 strain. The chemical controlresulted in about 19% of the larvae present on the treated filter paper.Table V shows similar results. The results show that the RTI545 strainwas unexpectedly superior to the chemical insecticide at repelling theinsects from the corn seed, but did not kill the insects.

Table VI compares the repellent effect on SCRW of kanosamine and B.thuringiensis strains RTI545 and FD30, showing that repellent behaviorof RTI545 against insects may be due to production of kanosamine.

EXAMPLE 7 shows the repellent and egg-hatching inhibition of root-knotnematodes exposed to RTI545 supernatent, compared to kanosamine. Theresults summarized in Tables VII and VIII suggest that the effect ofRTI545 against nematodes does not appear to be caused by production ofkanosamine.

EXAMPLE 8 describes the positive effect on growth and yield in field andgreenhouse trials under insect pressure by treating corn and soybeanseed with spores of B. thuringiensis RTI545. The effects on growth,yield, and control of corn pests, wireworm and seed maggot, weremeasured in field trials in Wisconsin. Additional experiments wereperformed in the greenhouse to measure the effect on early plant growthin the presence of wireworm.

In a field trial experiment, corn seeds were treated with slurriescontaining: 1) chemical fungal control treatment comprising MAXIM+APRONXL (referred to as “FC”); 2) FC+ the insecticide bifenthrin 0.125mg/seed; 3) FC+PONCHO 1250 (clothianidin 1.25 mg/seed) and VOTIVO(Bacillus firmus 1-1582); 4) FC+PONCHO 250 (clothianidin 0.25 mg/seed);5) FC+PONCHO 500 (clothianidin 0.5 mg/seed) and VOTIVO (Bacillus firmus1-1582) and; 6) FC+bifenthrin (0.125 mg/seed)+spores of B. thuringiensisRTI545. The treated corn seeds were planted in separate field trials inWisconsin in soil infested wireworm and seed maggot. The results areshown below in Table IX. Inclusion of the B. thuringiensis RTI545 incombination with the insecticide bifenthrin resulted in significantimprovements in percent emergence, plant stand, vigor and control ofboth wireworm and seed maggot over seeds treated with bifenthrin alone.In addition, the results of the combination of B. thuringiensis RTI545and bifenthrin were statistically equivalent to the product PONCHO 1250VOTIVO at controlling wireworm and showed an improvement over thisproduct in controlling seed maggot. These data indicate that corn seedtreatment with a combination of B. thuringiensis RTI545 and a chemicalinsecticide such as bifenthrin significantly improves insect controlover inclusion of chemical insecticide alone and is superior tocommercially available products for some types of insect control.

In a second field trial, corn seeds were treated with the same slurriesas the first trial containing: 1) chemical control MAXIM+APRON XL(referred to as “FC”); 2) FC+Bifenthrin; 3) FC+PONCHO 1250 VOTIVO; 4)FC+PONCHO 250; and 5) FC+Bifenthrin+spores of RTI545. The treated cornseed were planted in separate field trials in Wisconsin with wirewormpresent but without seed maggot. Damage of corn roots from wirewormfeeding were rated 41 days after planting. The results are shown belowin Table X and show similar results to the previous trial. Specifically,inclusion of the B. thuringiensis RTI545 in combination with theinsecticide bifenthrin resulted in significant improvements in percentemergence, plant stand, vigor and control of wireworm over seeds treatedwith bifenthrin alone. In addition, the results of the combination of B.thuringiensis RTI545 and bifenthrin were statistically equivalent orsuperior to the product PONCHO 1250 VOTIVO at controlling wireworm.

The average yield in the corn field trials after seed treatment with acombination of chemical insecticide and spores of RTI545 as compared toPONCHO VOTIVO was also determined. The results are shown below in TableXI. Inclusion of the B. thuringiensis RTI545 in combination with theinsecticide bifenthrin resulted in significant improvements in yield ascompared to seeds treated with bifenthrin alone. In addition, thecombination of RTI545 and bifenthrin outperformed both PONCHO 500 VOTIVOand PONCHO 1250 VOTIVO by increasing yield 13 bushels/acre (from 180.5to 193.7 and from 185.5 to 193.7 bushels/acre, respectively)representing a 6.8% and 4.2% increase in grain yield, respectively.These data indicate that corn seed treatment with a combination ofRTI545 and a chemical insecticide such as bifenthrin significantlyimproves yield over inclusion of chemical insecticide alone, and reducesthe need for in-furrow application of larger quantities of chemicalinsecticides to control damage by insects.

The effect on growth under insect pressure by treating corn seed withspores of RTI545 was further evaluated. In a set of greenhouse studies,corn seeds were first treated with the seed treatment slurries asdescribed as follows and then planted in soil infested with the pestwireworm (10 wireworms per pot with one seed), along with a control setwhere the soil did not contain wireworm. The seed treatment slurrieswere as follows: 1) chemical control MAXIM+APRON XL (referred to as“FC”); 2) FC; 3) FC+Bifenthrin (0125 mg/seed for all treatmentstreated); 4) FC+Bifenthrin+RTI545 5.0×10⁶; 5) FC+Bifenthrin+RTI5455.0×10⁶ heat-treated; 6) FC+Bifenthrin+RTI545 1.0×10⁶; 7) FC+RTI5455.0×10⁶; and 8) FC+PONCHO 1250. The treated seeds were evaluated forpercent emergence.

The results are shown below in Table XII. Inclusion of the B.thuringiensis RTI545 in combination with the insecticide bifenthrinresulted in 100% emergence, which was an improvement over inclusion ofbifenthrin alone and provided results equivalent to the control withoutwireworm and the FC+PONCHO 1250 chemical treatment. Wireworm feedingprunes roots causing corn plant stunting and RTI545 alone or Bifenthrinwith RTI545 reduced plant stunting in surviving plants. RTI545 aloneexhibited activity on preventing plant loss but was inferior toinsecticide bifenthrin in providing early protection against stunting.However, RTI545 was more effective in preventing plant stunting as theplants grew (data not shown). These data indicate that inclusion ofspores of RTI545 in corn seed treatment, alone or in combination with achemical insecticide such as bifenthrin, significantly improves planthealth in the presence of the insect pest wireworm.

Experiments were performed to determine the effect on yield by treatingsoybean seed with a standard fungicidal combination of chemical activeingredients in addition to spores of B. thuringiensis RTI545, incombination with a chemical insecticide. The experiments were performedas described below. In the experiment, soybean seeds were mixed with asolution containing: 1) chemical control fludioxonil/TPM/mefenoxam(“FC”); 2) FC+insecticide thiamethoxam; and 3) FC+thiamethoxam+spores ofB. thuringiensis RTI545. The treated soybean seeds were planted at threesites (N=3) that had wireworm infestation, and the yield was analyzed.The results are shown below in Table XIII. Inclusion of the RTI545spores in combination with the thiamethoxam resulted in significantimprovements in yield as compared to seeds treated with thiamethoxamalone. These data indicate that soybean seed treatment with acombination of RTI545 and a chemical insecticide significantly improvesyield over inclusion of insecticide alone, and reduces the need forin-furrow application of larger quantities of chemical insecticides tocontrol damage by insects.

EXAMPLE 9 describes the ability of the Bacillus thuringiensis RTI545strain to reduce the nematode infestation in soybean and potato. Agreenhouse study was performed with soybean plants potted in soilinfected with Southern root-knot nematodes (Meloidogyne incognita) fromseed treated with and without RTI545 cells. Seed treatment includedproducts PONCHO VOTIVO (BAYER CROPSCIENCE LP; A.I.=40.3% clothianidin,8.1% Bacillus firmus 1-1582) and AVICTA COMPLETE (SYNGENTA; A.I.=11.7%thiamethoxam, 10.3% abamectin, 2.34% thiabendazole, 0.3% fludioxonil,0.23% mefenoxam, 0.12% azoxystrobin). The results are shown in Table XIVbelow. At 63 days after initiation, there was no statistical differencein the number of nematode eggs/pot for the seed treated with RTI545cells and seed treated with the chemical combination AVICTA COMPLETE.However, the number of nematode eggs/pot for the seed treated withRTI545 cells was less than that for the seed treated with PONCHO VOTIVO.This demonstrates the positive effect on nematode control on soybeanprovided by Bacillus thuringiensis RTI545, providing equivalent tosuperior control as compared to commercially available biological pluschemical- and chemical active-based products.

A greenhouse study was performed with potato plants potted in soilnaturally infected with Globodera sp. nematodes to determine the effectof treatment of the soil with Bacillus thuringiensis RTI545 cells.Potatoes (nematode sensitive variety “Bintje”) were planted in soilinfected with Globodera sp. (Control) and enhanced with 10⁹ cfu sporesper liter of soil of RTI545. The results are shown in the graph in FIG.5. Additional soil treatments were included in the study: VYDATE product(DUPONT; A.I.=Oxamyl [Methoyl N′N′-dimethyl-N-[(methylcarbamoyl)oxyl)oxy]-1-thiooxamimidate), BIOACT product (BAYERCROPSCIENCES LP; A.I.=Paecilomyces lilacinus strain 251), CAREX product(NUFARM, pyridaben), and Bacillus thuringiensis spp. kurstaki HD-1). Theproducts were applied at the rates designated on the product labels. Ascan be seen in FIG. 5, the number of cysts per gram of root biomass forthe soil treated with RTI545 cells was significantly reduced compared toall of the treatments including the products containing chemical activeingredients. This demonstrates the positive effect on nematode controlfor potato provided by Bacillus thuringiensis RTI545, providing superiorcontrol as compared to commercially available biological- and chemicalactive-based products.

EXAMPLE 10 describes experiments performed to investigate the effect onemergence, root disease, and yield in cotton in the presence ofRhizoctonia disease pressure when seeds were treated with the RTI545strain in addition to chemical active agents for pathogen control.

Specifically, an experiment in cotton was set up as follows: 1) seed wasuntreated (UTC); 2) seed was treated with a base combination offludioxonil+mefenoxam+imidacloprid according to manufacturer label(referred to as “B”); 3) seed was treated with base plus 5×10⁵ cfu/seedof RTI545 (B+RTI545); and 4) seed was treated with base plus VIBRANCE(active ingredient sedaxane; SYNGENTA CROP PROTECTION, INC) according tolabel instructions (B+VIBRANCE). Field trials were performed in Georgia.The trials were inoculated with Rhizoctonia by mixing the dried inoculumwith the seed at the time of planting to a prescribed rate to provideinfection when the seed commenced to grow. The average percent cottonemergence is presented in Table XV below.

The results in Table XV show that treating with RTI545 spores inaddition to the base resulted in significant improvement in percentemergence over that of the chemical base alone. In addition, treatmentwith RTI545 performed as well as the base plus commercial productVIBRANCE with chemical active. Thus, seed treatment with RTI545 canprovide significant improvement in emergence even under conditions ofsevere pathogen pressure.

EXAMPLE 11 describes experiments to investigate the effect on growth andyield under insect pressure by wheat seed treated with spores of B.thuringiensis RTI545 and seeds treated with RTI545 plus chemical activeagents for pathogen control. More specifically, the effects on growth,yield, and control of wheat pests, wireworm and white grub, weremeasured in field trials in Wisconsin. Wheat seeds were treated withslurries containing: 1) chemical fungicide basedifenoconazole/tebuconazole/TPM/mefenoxan (referred to as “FC”); 2)FC+spores of B. thuringiensis RTI545 (RTI545 1×10⁶ cfu/g seed); 3)FC+bifenthrin (20 g/seed); 4) FC+bifenthrin (20 g/seed)+RTI545 1×10⁶cfu/g seed; 5) FC+bifenthrin (50 g/seed); and 6) FC+bifenthrin (50g/seed)+RTI545 1×10⁶ cfu/g seed. The results are shown below in TableXVI. Seed treatment with each of the B. thuringiensis RTI545 spores, andtreatment with the RTI545 spores with either the fungicide base alone orin combination with the insecticide, bifenthrin, resulted in significantimprovements in percent emergence, vigor, control of wireworm and whitegrub, and yield. In every case tested, inclusion of the RTI545 spores inthe wheat seed treatment resulted in significant improvements in growth,vigor, pest control, and yield.

EXAMPLE 12 shows the impact of RTI545 soybean seed treatment on growthand yield against the Rhizoctonia fungal pathogen. Table XVII shows acomparison of RTI545 added to a base chemical seed treatment compared tothe base treatment alone and to the base treatment plus another chemicalactive ingredient. Addition of RTI545 to the base chemical treatmentresulted in significant increases in stand, vigor and yield compared tothe chemical base.

EXAMPLE 13 shows the impact of RTI545 corn seed treatment on growth andyield against wireworms and seed maggots. Table XVIII shows that RTI545provides some protection and enhances insecticidal protection againsttheses pests when added to chemical insecticides such as chlothinidin,bifenthrin and chlorantraniliprole.

EXAMPLE 14 shows that RTI545 is effective in in-furrow applicationsagainst corn wireworm and corn rootworm, resulting in increased yieldand reduced root damage, alone and especially in combination with thechemical insecticide bifentrhrin, as shown in Tables XIX and XX.

EXAMPLE 15 shows the impact of RTI545 peanut seed treatment on growthand yield against the Rhizoctonia fungal pathogen. Table XXI shows acomparison of RTI545 added to a base chemical seed treatment compared tothe base treatment alone. Addition of RTI545 to the base chemicaltreatment resulted in significant increases in stand, vigor and yieldcompared to the chemical base.

EXAMPLE 16 presents greenhouse assays of RTI545 corn seed treatmentagainst lesion nematodes. Table XXII shows a comparison of RTI545 addedto a base fungicide chemical seed treatment compared to the basetreatment alone and the base treatment plus PONCHO/VOTIVO.

RTI545 plus the base treatment provided superior root length increaseover the untreated compared to that provided by the base treatment plusPONCHO/VOTIVO. Table XXIII shows that RTI545+ base treatment providedsuperior reduction in penetration and fresh top weight compared to thebase treatment and the base treatment+PONCHO/VOTIVO. This table alsoshows results when RT545 is combined with other biological controlagents.

Example 17 presents soil drench assays of RTI545 soybean seed treatmentagainst soybean cyst nematodes. Table XXIV shows a reduction of cystscompared to the untreated control.

EXAMPLES 18 and 19 show suspension concentrate formulations of RTI545(Table XXV) and RTI545 plus bifenthrin (Tables XXVI and XXVII). TableXXVIII shows that the spores in an SC formulation remained stable overtwo weeks of storage at elevated temperature

In one embodiment of the present disclosure, a composition is providedthat includes a biologically pure culture of Bacillus thuringiensisRTI545 deposited as ATCC No. PTA-122161, or a mutant thereof having allthe identifying characteristics thereof, for application to a plant forone or both of benefiting plant growth or conferring protection againsta plant pest in a susceptible plant.

In another embodiment, a method is provided for one or both ofbenefiting growth of a plant or conferring protection against a plantpest in a susceptible plant, the method including delivering acomposition comprising a biologically pure culture of Bacillusthuringiensis RTI545 deposited as ATCC No. PTA-122161, or a mutantthereof having all the identifying characteristics thereof to: a plant,plant part, seed of the plant, roots of the plant, soil or growth mediumsurrounding the plant or the seed of the plant, or soil or growth mediumbefore planting the plant or sowing seed of the plant, in an amountsuitable to benefit the plant growth and/or to confer protection againstthe plant pest in the susceptible plant.

In the compositions and methods of the present disclosure, the growthbenefit of the plant and/or the conferred protection can be exhibited byimproved seedling vigor, improved root development, improved plantgrowth, improved plant health, increased yield, improved appearance,improved resistance to plant pests, reduced pathogenic infection, or acombination thereof.

The compositions and methods of the present invention are beneficial toa wide range of plants including, but not limited to, monocots, dicots,cereals such as corn, sweet corn, popcorn, seed corn, silage corn, fieldcorn, rice, wheat, barley, sorghum, asparagus, berries such asblueberry, blackberry, raspberry, loganberry, huckleberry, cranberry,gooseberry, elderberry, currant, caneberry, bushberry, brassicavegetables such as broccoli, cabbage, cauliflower, brussels sprouts,collards, kale, mustard greens, kohlrabi, cucurbit vegetables such ascucumber, cantaloupe, melon, muskmelon, squash, watermelon, pumpkin,eggplant, bulb vegetables such as onion, garlic, shallots, citrus suchas orange, grapefruit, lemon, tangerine, tangelo, pummelo, fruitingvegetables such as pepper, tomato, ground cherry, tomatillo, okra,grape, herbs, spices, leafy vegetables such as lettuce, celery, spinach,parsley, radicchio, legumes or vegetables such as beans including greenbeans, snap beans, shell beans, soybeans, dry beans, garbanzo beans,lima beans, peas, chick peas, split peas, lentils, oil seed crops suchas canola, castor, coconut, cotton, flax, oil palm, olive, peanut,rapeseed, safflower, sesame, sunflower, soybean, pome fruit such asapple, crabapple, pear, quince, mayhaw, root, tuber and corm vegetablessuch as carrot, potato, sweet potato, cassava, beets, ginger,horseradish, radish, ginseng, turnip, stone fruit such as apricot,cherry, nectarine, peach, plum, prune, strawberry, tree nuts such asalmond, pistachio, pecan, walnut, filberts, chestnut, cashew, beechnut,butternut, macadamia, kiwi, banana, (blue) agave, grass, turf grass,ornamental plants, poinsettia, hardwood cuttings such as chestnut, oak,maple, sugarcane, and sugarbeet. In one or more embodiments, the plantcan include corn, soybean, potato, cotton, tomato, pepper, cucurbits,sugarcane, peanut or wheat; or soybean, cotton, wheat, corn or potato.

In the compositions and methods of the present invention, the plantdamage can be caused by a wide variety of plant pests including, forexample, but not limited to, a plant insect pest, such as a WesternPlant Bug (WPB) Lygus hesperus, Coleoptera sp., Diabrotica sp.,including Western (D. virgifera), Southern (D. undecimpunctata) andNorthern (D. barberi) corn rootworm), D. balteata, and D. longicornis,Melanotus spp. (including corn wireworm, Melanotus communis andMelanotus cribulosus), Phyllophaga spp. (including white grubs,wireworms, false wireworms and Phyllophaga rugosa), Limonius spp.(sugarbeet wireworms and Limonius agronus), Agriotes spp. (includingwheat wireworms Agriotes mancus, corn wireworms, white grubs and Seedmaggots), Lepidoptera sp., Peridroma spp. (including variegatedcutworm), Euxoa spp. (including army cutworm), Agrotis spp. (includingblack cutworm Agrotis ipsilon), Diptera sp., Hylemya spp. (includingseedcorn maggot Delia platura Meigen and Hylemya cilicrura), Tetanopsspp. (including sugarbeet root maggot), Homoptera sp., Pemphigus sp.(including sugarbeet root aphid, cutworm, and white grub), Aphis spp.(including corn root aphid), seed-corn beetle Agonoderus lecontei,Feltia subgothica, or combinations thereof. Notable insects includeWestern plant bug, Southern corn rootworm, corn wireworm, seedcornmaggot, seed maggot, wheat wireworm and white grub.

In the compositions and methods of the present invention, the plant pestcan be, for example, but not limited to, a plant pathogenic nematode, areniform nematode, Rotlyenchulus spp., dagger nematode, Xiphinema spp.,lance nematode, Hoplolaimus spp., pin (lesion) nematode, Paratylenchusspp., ring nematode, Criconemoides spp., root-knot nematode, Meloidogynespp., sheath nematode, Hemicycliophora spp., spiral nematode,Helicotylenchus spp., stubbyroot nematode, Trichodorus spp., cystnematode, Heterodera spp. and Globodera spp., sting nematode,Belonolaimus spp., stunt nematode, and Tylenchorhynchus spp., burrowingnematode, Radopholus spp. or combinations thereof. Notable nematodesinclude root-knot nematodes (Meloidogyne spp.), lesion nematodes,(Paratylenchus spp.) and cyst nematodes (Heterodera spp. and Globoderaspp.).

In the compositions and methods of the present invention, the plant pestcan be characterized by, for example, but not limited to, a plant fungalpathogen or a plant bacterial pathogen, such as a rust fungus, aBotrytis spp., an Erwinia spp., a Dickeya spp., an Agrobacterium spp., aXanthomonas spp., a Xylella spp., a Candidatus spp., a Fusarium spp., aSclerotinia spp., a Cercospora/Cercosporidium spp., an Uncinula spp., aPodosphaera spp. (Powdery Mildew), a Phomopsis spp., an Alternaria spp.,a Pseudomonas spp., a Phytophthora spp., a Phakopsora spp., anAspergillus spp., a Uromyces spp., such as Uromyces appendiculatus, aCladosporium spp., a Rhizopus spp., a Penicillium spp., a Rhizoctoniaspp., Macrophomina phaseolina, a, a Mycosphaerella spp., a Magnaporthespp., such as Magnaporthe oryzae or Magnaporthe grisea, a Moniliniaspp., a Colletotrichum spp., a Diaporthe spp., a Corynespora spp., aGymnosporangium spp., a Schizothyrium spp., a Gloeodes spp., aBotryosphaeria spp., a Neofabraea spp., a Wilsonomyces spp., aSphaerotheca spp., a Erysiphe spp., a Stagonospora spp., a Pythium spp.,a Venturia spp., a Ustilago spp., a Claviceps spp., a Tilletia spp., aPhoma spp., Cocliobolus sativus, Gaeumanomyces gaminis, a Rhynchosporiumspp., a Biopolaris spp., and a Helminthosporium spp., or combinationsthereof.

Notable fungal pathogens include Aspergillus flavus, Botrytis spp. suchas Botrytis cinerea, Fusarium spp., such as Fusarium colmorum, Fusariumoxysporum or Fusarium virguliforme, Phytophthora spp. such asPhytophthora capsici, Rhizoctonia spp. such as Rhizoctonia solani,Magnaporthe spp., such as Magnaporthe grisea and Magnaporthe oryzae, andPythium spp. such as Pythium aphanidermatum and Pythium sylvatium,Monilinia spp. such as Monilinia fructicola, Colletotrichum spp., suchas Colletotrichum gloeosporioides (sexual stage Glomerella cingulata)i.e. anthracnose, Sclerotinia spp., such as Sclerotinia sclerotiorum andSclerotinia homeocarpa; more notably Rhizoctonia spp. Notable bacterialpathogens include Erwinia spp. such as Erwinia amylovora.

In the compositions and methods of the present disclosure, thecompositions including the RTI545 strain can be in the form of a liquid,a suspension concentrate, an oil dispersion, a dust, a dry wettablepowder, a spreadable granule, or a dry wettable granule. In embodiments,the Bacillus thuringiensis RTI545 can be present in the composition at aconcentration of from about 1.0×10⁸ CFU/ml to about 1.0×10¹² CFU/ml. Inembodiments, the Bacillus thuringiensis RTI545 can be present in anamount of from about 1.0×10⁸ CFU/g to about 1.0×10¹² CFU/g. The Bacillusthuringiensis RTI545 can be in the form of spores or vegetative cells.

In the compositions and methods of the present disclosure, thecomposition including the RTI545 strain can include one or a combinationof adjuvants including for example a carrier, a binder, a surfactant, adispersant, or a yeast extract. The carrier, binder, surfactant,dispersant, and/or yeast extract are included to improve the propertiesof the composition for use in benefiting plant growth and or conferringprotection against plant pests, the properties including one or more ofimproved handling properties, improved wettability, improvedflowability, improved adhesion to seed, improved stability of the RTI545strain, and improved activity of the RTI545 strain after delivery orapplication to the plant seed, roots, or soil. The yeast extract can bedelivered at a rate for benefiting plant growth ranging from about 0.01%to 0.2% w/w.

The compositions including the RTI545 strain can be in the form of aplanting matrix. The planting matrix can be in the form of a pottingsoil mixture.

In the compositions and methods of the present disclosure, thecomposition can further include one or a combination of an additionalagricultural agent, such as an insecticide, fungicide, nematicide,bacteriocide, biostimulant, herbicide, plant extract, microbial extract,plant growth regulator, fertilizer or crop nutrient product present inan amount suitable to benefit the plant growth and/or to conferprotection against the plant pest in the susceptible plant. In oneembodiment, the composition including the biologically pure culture ofBacillus thuringiensis RTI545 and the one or a combination of theinsecticide, fungicide, nematicide, bacteriocide, biostimulant,herbicide, plant extract, microbial extract, plant growth regulator,fertilizer or crop nutrient product, are formulated together. Any of theadditional agricultural agents may be a biological agent or a chemicalagent. In other embodiments the composition comprising the RTI545 strainis formulated separately from the additional agricultural agent, whichmay also be formulated, and then mixed with the additional agriculturalagent, such as in a tank mix.

The fertilizer can be a liquid fertilizer. The term “liquid fertilizer”refers to a fertilizer in a fluid or liquid form containing variousratios of nitrogen, phosphorous and potassium (for example, but notlimited to, 5 to 15%, such as 10%, of nitrogen, 20 to 50%, such as 34%,of phosphorous and 0 to 15%, such as 0%, of potassium) and optionallysecondary nutrients and/or micronutrients, commonly known as starterfertilizers that are high in phosphorus and promote rapid and vigorousroot growth.

As used herein, the term “biostimulant” refers to a substance ormicroorganism applied to plants with the aim of enhancing nutrientuptake, nutrition efficiency, and/or abiotic stress tolerance to improvecrop vigor, yields and/or crop quality traits such as nutritionalcontent, appearance and shelf-life, regardless of its nutrient content.Biostimulants operate through different mechanisms than fertilizers anddo not have direct action against pest or diseases.

In one embodiment, the composition including the biologically pureculture of Bacillus thuringiensis RTI545 further comprises the chemicalinsecticide bifenthrin.

Of note are mixtures of the Bacillus thuringiensis RTI545 strain withother biocontrol strains including other Bacillus thuringiensis strainssuch as Bacillus thuringiensis subsp. aizawai or Bacillus thuringiensissubsp. kurstaki, Bacillus subtilis strains such as CH201 or QST713 orMBI600 or RTI477, Bacillus licheniformis strains such as CH200 orRTI184, Bacillus velezensis strains such as RTI301, Bacillus subtilisvar. amyloliquefaciens FZB24, or Bacillus amyloliquefaciens D747, orcombinations thereof.

Mixtures of microbial strains, including RTI545, can be used to enhanceactivity against specific target pests, but can also be used to enhancethe spectrum of utility. As an example, combining a strain with strongactivity against soil insects with other strains that have strongactivity against nematodes, fungi, or strong plant growth benefits canbe combined. Furthermore, such mixtures of strains can also be combinedwith synthetic (chemical) pesticides for added benefits.

In one embodiment, the composition including the biologically pureculture of Bacillus thuringiensis RTI545 further comprises a strainpreviously identified as Bacillus amyloliquefaciens RTI301 deposited asATCC No. PTA-121165 (See US2016/0186273). This strain has recently beenreclassified as a Bacillus velezensis strain. In the remainder of thisspecification, the strain deposited as ATCC No. PTA-121165 will bereferred to as “RTI301” or “Bacillus velezensis RTI301”. In oneembodiment, the combination of the RTI545 and the RTI301 strains extendsthe benefit to plant growth and protection against plant pests bywidening and increasing the temperature range in which one or both ofthe strains provide maximum protection against plant pests, includingplant fungal pathogens.

In other embodiments, the composition including the biologically pureculture of Bacillus thuringiensis RTI545 further comprises abiologically pure culture of a Bacillus licheniformis CH200 deposited asDSM 17236, or a mutant thereof having all the identifyingcharacteristics thereof; a biologically pure culture of a Bacillussubtilis CH201 deposited as DSM 17231, or a mutant thereof having allthe identifying characteristics thereof; a biologically pure culture ofa Bacillus subtilis RTI477 deposited as ATCC No. PTA-121167, or a mutantthereof having all the identifying characteristics thereof; abiologically pure culture of a Bacillus amyloliquefaciens D747 straindeposited as FERM BP-8234; a Bacillus licheniformis RTI184 deposited asATCC No. PTA-121722, or a mutant thereof having all the identifyingcharacteristics thereof or any combination thereof, includingcombinations also comprising RTI301.

In one embodiment, the composition including the biologically pureculture of Bacillus thuringiensis RTI545 further comprises the chemicalinsecticide, bifenthrin, and the composition is delivered in combinationwith a liquid fertilizer to: a plant, plant part, seed of the plant,roots of the plant, soil or growth medium surrounding the plant or theseed of the plant, or soil or growth medium before planting the plant orsowing seed of the plant. Examples of using the insecticide, bifenthrin,in combination with a liquid fertilizer to benefit plant growth aredescribed, for example, in WO 2016/108972 A1, which is hereinincorporated by reference in its entirety.

In another embodiment, a method is provided for one or both ofbenefiting growth of a plant or conferring protection against a plantpest in a susceptible plant, the method comprising: delivering to aplant, plant part, seed of the plant, roots of the plant, soil or growthmedium surrounding the plant or the seed of the plant, or soil or growthmedium before planting the plant or sowing seed of the plant, acombination of: a composition comprising a biologically pure culture ofBacillus thuringiensis RTI545 deposited as ATCC No. PTA-122161, or amutant thereof having all the identifying characteristics thereof in anamount suitable to benefit the plant growth and/or to confer protectionagainst the plant pest in the susceptible plant; and one or acombination of additional agricultural agents such as an insecticide,fungicide, nematicide, bacteriocide, herbicide, plant extract, plantgrowth regulator, or fertilizer as described herein in an amountsuitable to benefit the plant growth and/or to confer protection againstthe plant pest in the susceptible plant. Any of the additionalagricultural agents may be a biological agent or a chemical agent. Inthis embodiment, the composition including the biologically pure cultureof Bacillus thuringiensis RTI545 and the one or a combination ofadditional agricultural agent(s) as described herein, are deliveredseparately to the susceptible plant, rather than from a singleformulation.

In one embodiment, the composition including the biologically pureculture of Bacillus thuringiensis RTI545 is delivered in combinationwith a liquid fertilizer to: plant, plant part, seed of the plant, rootsof the plant, soil or growth medium surrounding the plant or the seed ofthe plant, or soil or growth medium before planting the plant or sowingseed of the plant.

In one embodiment, the composition including the biologically pureculture of Bacillus thuringiensis RTI545 is delivered in combinationwith the chemical insecticide, bifenthrin, and with the liquidfertilizer to: plant, plant part, seed of the plant, roots of the plant,soil or growth medium surrounding the plant or the seed of the plant, orsoil or growth medium before planting the plant or sowing seed of theplant.

In one embodiment, a plant seed is provided that is coated with acomposition comprising an additional biological agricultural agent, suchas: spores of a biologically pure culture of Bacillus velezensis RTI301deposited as ATCC No. PTA-121165, or a mutant thereof having all theidentifying characteristics thereof, present in an amount suitable tobenefit plant growth and/or to confer protection against a plant pest ina susceptible plant. The composition can include an amount of Bacillusvelezensis spores from about 1.0×10² CFU/seed to about 1.0×10⁹ CFU/seed.

The coated seed compositions of the present invention are beneficial toa wide range of plant seeds including, but not limited to, the seed ofmonocots, dicots, cereals such as corn, sweet corn, popcorn, seed corn,silage corn, field corn, rice, wheat, barley, sorghum, asparagus,berries such as blueberry, blackberry, raspberry, loganberry,huckleberry, cranberry, gooseberry, elderberry, currant, caneberry,bushberry, brassica vegetables such as broccoli, cabbage, cauliflower,brussels sprouts, collards, kale, mustard greens, kohlrabi, cucurbitvegetables such as cucumber, cantaloupe, melon, muskmelon, squash,watermelon, pumpkin, eggplant, bulb vegetables such as onion, garlic,shallots, citrus such as orange, grapefruit, lemon, tangerine, tangelo,pummelo, fruiting vegetables such as pepper, tomato, ground cherry,tomatillo, okra, grape, herbs, spices, leafy vegetables such as lettuce,celery, spinach, parsley, radicchio, legumes or vegetables such as beansincluding green beans, snap beans, shell beans, soybeans, dry beans,garbanzo beans, lima beans, peas, chick peas, split peas, lentils, oilseed crops such as canola, castor, coconut, cotton, flax, oil palm,olive, peanut, rapeseed, safflower, sesame, sunflower, soybean, pomefruit such as apple, crabapple, pear, quince, mayhaw, root, tuber andcorm vegetables such as carrot, potato, sweet potato, cassava, beets,ginger, horseradish, radish, ginseng, turnip, stone fruit such asapricot, cherry, nectarine, peach, plum, prune, strawberry, tree nutssuch as almond, pistachio, pecan, walnut, filberts, chestnut, cashew,beechnut, butternut, macadamia, kiwi, banana, (blue) agave, grass, turfgrass, ornamental plants, poinsettia, hardwood cuttings such aschestnuts, oak, maple, sugarcane, and sugarbeet. In one or moreembodiments, the plant seed can include corn, soybean, potato, cotton,tomato, pepper, cucurbits, sugarcane, peanut or wheat; or soybean,cotton, wheat, corn or potato.

In one embodiment of the plant seed coated with the composition, thecomposition further comprises one or a combination of additionalagricultural agent(s) as described herin present in an amount suitableto benefit plant growth and/or to confer protection against the plantpest in the susceptible plant.

In one embodiment, a method is provided for one or both of benefitinggrowth of a plant or conferring protection against a plant pest in asusceptible plant, the method comprising: planting a seed of the plant,wherein the seed has been coated with a composition comprising abiologically pure culture of Bacillus thuringiensis RTI545 deposited asATCC PTA-122161, or a mutant thereof having all the identifyingcharacteristics thereof, wherein growth of the plant from the seed isbenefited and/or protection against the plant pest is conferred.

In one embodiment, the method further includes delivering a liquidfertilizer to the coated seed of the plant, soil or growth mediumsurrounding the coated seed of the plant, or soil or growth mediumbefore planting the coated seed of the plant.

In one embodiment of the method, the plant seed is coated with thecomposition further comprising an additional biological agriculturalagent, such as Bacillus velezensis RTI301 deposited as ATCC No.PTA-121165. In one embodiment, the combination of the RTI545 and theRTI301 strains extends the benefit to plant growth and protectionagainst plant pests by widening and increasing the temperature range inwhich one or both of the strains provide maximum protection againstplant pests, including plant fungal pathogens.

In one embodiment, the plant seed is coated with the composition furthercomprising a chemical insecticide, and the method further includesdelivering a liquid fertilizer to the coated seed of the plant, soil orgrowth medium surrounding the coated seed of the plant, or soil orgrowth medium before planting the coated seed of the plant. In oneembodiment, the chemical insecticide is bifenthrin.

In embodiments herein comprising coated seeds, the term “seed” refersnot only to true seeds but also other plant parts for propagating theplant such as seedlings, transplants, cuttings (e.g. stems, roots,leaves, and the like), spores, setts (e.g. of sugarcane), bulbs, corms,rhizomes, tubers, or portions thereof, or other plant tissue from whicha complete plant can be obtained.

In the compositions and methods of the present disclosure, thecomposition can include a fungicide. The fungicide can include anextract from Lupinus albus. In one or more embodiments, the fungicidecan include a BLAD polypeptide. The BLAD polypeptide can be a fragmentof the naturally occurring seed storage protein from sweet lupine(Lupinus albus) that acts on susceptible fungal pathogens by causingdamage to the fungal cell wall and disrupting the inner cell membrane.The compositions can include about 20% of the BLAD polypeptide.

In addition, in one or more embodiments, suitable insecticides,herbicides, fungicides, and nematicides of the compositions and methodsof the present invention can include the following:

Insecticides: A0) various insecticides, including agrigata,al-phosphide, amblyseius, aphelinus, aphidius, aphidoletes, artimisinin,autographa californica NPV, azocyclotin, Bacillus subtilis, Bacillusthuringiensis subsp. aizawai, Bacillus thuringiensis subsp. kurstaki,Bacillus thuringiensis, Beauveria, Beauveria bassiana, betacyfluthrin,biologicals, bisultap, brofluthrinate, bromophos-e, bromopropylate,Bt-Corn-GM, Bt-Soya-GM, capsaicin, cartap, celastrus-extract,chlorantraniliprole, chlorbenzuron, chlorethoxyfos, chlorfluazuron,chlorpyrifos-e, cnidiadin, cryolite, cyanophos, cyantraniliprole,cyclaniliprole, cyhalothrin, cyhexatin, cypermethrin, dacnusa, DCIP,dichloropropene, dicofol, diglyphus, diglyphus+dacnusa, dimethacarb,dithioether, dodecyl-acetate, emamectin, encarsia, EPN, eretmocerus,ethylene-dibromide, eucalyptol, fatty-acids, fatty-acids/salts,fenazaquin, fenobucarb (BPMC), fenpyroximate, flubrocythrinate,flufenzine, flupyradifurone, formetanate, formothion, furathiocarb,gamma-cyhalothrin, garlic-juice, granulosis-virus, harmonia, heliothisarmigera NPV, inactive bacterium, indol-3-ylbutyric acid, iodomethane,iron, isocarbofos, isofenphos, isofenphos-m, isoprocarb, isothioate,kaolin, lindane, liuyangmycin, matrine, mephosfolan, metaldehyde,metarhizium-anisopliae, methamidophos, metolcarb (MTMC), mineral-oil,mirex, m-isothiocyanate, monosultap, myrothecium verrucaria, naled,neochrysocharis formosa, nicotine, nicotinoids, oil, oleic-acid,omethoate, orius, oxymatrine, paecilomyces, paraffin-oil, parathion-e,pasteuria, petroleum-oil, pheromones, phosphorus-acid, photorhabdus,phoxim, phytoseiulus, pirimiphos-e, plant-oil, plutella xylostella GV,polyhedrosis-virus, polyphenol-extracts, potassium-oleate, profenofos,prosuler, prothiofos, pyraclofos, pyrethrins, pyridaphenthion,pyrimidifen, pyriproxifen, quillay-extract, quinomethionate, rape-oil,rotenone, saponin, saponozit, sodium-compounds, sodium-fluosilicate,starch, steinernema, streptomyces, sulfluramid, sulphur, tebupirimfos,temephos, tetradifon, tetraniliprole, thiofanox, thiometon, transgenics(e.g., Cry3Bb1), triazamate, trichoderma, trichogramma, triflumuron,verticillium, vertrine, isomeric insecticides (e.g., kappa-bifenthrin,kappa-tefluthrin), dichoromezotiaz, broflanilide, pyraziflumid; A1) theclass of carbamates, including aldicarb, alanycarb, benfuracarb,carbaryl, carbofuran, carbosulfan, methiocarb, methomyl, oxamyl,pirimicarb, propoxur and thiodicarb; A2) the class of organophosphates,including acephate, azinphos-ethyl, azinphos-methyl, chlorfenvinphos,chlorpyrifos, chlorpyrifos-methyl, demeton-S-methyl, diazinon,dichlorvos/DDVP, dicrotophos, dimethoate, disulfoton, ethion,fenitrothion, fenthion, isoxathion, malathion, methamidaphos,methidathion, mevinphos, monocrotophos, oxymethoate, oxydemeton-methyl,parathion, parathion-methyl, phenthoate, phorate, phosalone, phosmet,phosphamidon, pirimiphos-methyl, quinalphos, terbufos,tetrachlorvinphos, triazophos and trichlorfon; A3) the class ofcyclodiene organochlorine compounds such as endosulfan; A4) the class offiproles, including ethiprole, fipronil, pyrafluprole and pyriprole; A5)the class of neonicotinoids, including acetamiprid, clothianidin,dinotefuran, imidacloprid, nitenpyram, thiacloprid and thiamethoxam; A6)the class of spinosyns such as spinosad and spinetoram; A7) chloridechannel activators from the class of mectins, including abamectin,emamectin benzoate, ivermectin, lepimectin and milbemectin; A8) juvenilehormone mimics such as hydroprene, kinoprene, methoprene, fenoxycarb andpyriproxyfen; A9) selective homopteran feeding blockers such aspymetrozine, flonicamid and pyrifluquinazon; A10) mite growth inhibitorssuch as clofentezine, hexythiazox and etoxazole; A11) inhibitors ofmitochondrial ATP synthase such as diafenthiuron, fenbutatin oxide andpropargite; uncouplers of oxidative phosphorylation such aschlorfenapyr; A12) nicotinic acetylcholine receptor channel blockerssuch as bensultap, cartap hydrochloride, thiocyclam and thiosultapsodium; A13) inhibitors of the chitin biosynthesis type 0 from thebenzoylurea class, including bistrifluron, diflubenzuron, flufenoxuron,hexaflumuron, lufenuron, novaluron and teflubenzuron; A14) inhibitors ofthe chitin biosynthesis type 1 such as buprofezin; A15) moltingdisruptors such as cyromazine; A16) ecdyson receptor agonists such asmethoxyfenozide, tebufenozide, halofenozide and chromafenozide; A17)octopamin receptor agonists such as amitraz; A18) mitochondrial complexelectron transport inhibitors pyridaben, tebufenpyrad, tolfenpyrad,flufenerim, cyenopyrafen, cyflumetofen, hydramethylnon, acequinocyl orfluacrypyrim; A19) voltage-dependent sodium channel blockers such asindoxacarb and metaflumizone; A20) inhibitors of the lipid synthesissuch as spirodiclofen, spiromesifen and spirotetramat; A21) ryanodinereceptor-modulators from the class of diamides, including flubendiamide,the phthalamide compounds(R)-3-Chlor-N1-{2-methyl-4-[1,2,2,2-tetrafluor-1-(trifluormethyl)ethyl]phenyl}-N2-(1-methyl-2-methylsulfonylethyl)phthalamidand(S)-3-Chlor-N1-{2-methyl-4-[1,2,2,2-tetrafluor-1-(trifluormethyl)ethyl]phenyl}-N2-(1-methyl-2-methylsulfonylethyl)phthalamid,chlorantraniliprole, cyclaniliprole and cyantraniliprole; A22) compoundsof unknown or uncertain mode of action such as azadirachtin,amidoflumet, bifenazate, fluensulfone, piperonyl butoxide, pyridalyl,sulfoxaflor; or A23) sodium channel modulators from the class ofpyrethroids, including acrinathrin, allethrin, bifenthrin, cyfluthrin,lambda-cyhalothrin, cypermethrin, alpha-cypermethrin, beta-cypermethrin,zeta-cypermethrin, deltamethrin, esfenvalerate, etofenprox,fenpropathrin, fenvalerate, flucythrinate, tau-fluvalinate, permethrin,silafluofen, tefluthrin and tralomethrin.

Of note are mixtures of the Bacillus thuringiensis RTI545 strain withother biocontrol strains including Bacillus subtilis strains such asCH201, Bacillus licheniformis strains such as CH200, other Bacillusthuringiensis strains such as Bacillus thuringiensis subsp. aizawai,Bacillus thuringiensis subsp. kurstaki, or combinations thereof forinsect control.

In the compositions and methods of the present disclosure, thecomposition can include a chemical insecticide. The chemical insecticidecan include a pyrethroid such as bifenthrin, tefluthrin,zeta-cypermethrin, cyfluthrin; an organophosphate such aschlorethoxyphos, chlorpyrifos, tebupirimphos, fiproles such as fipronil;neonicotinoids such as imidacloprid, thiamethoxam, clothianidin;diamides such as chlorantraniliprole, cyantraniliprole, cyclaniliprole;or mixtures thereof.

Preferred are mixtures of the Bacillus thuringiensis RTI545 strain withchemical insect control agents comprising chlorantraniliprole,chlorethoxyfos, chlorpyrifos-e, cyantraniliprole, cyclaniliprole,cypermethrin, dichloropropene, flupyradifurone, gamma-cyhalothrin,profenofos, tebupirimfos, tefluthrin, kappa-bifenthrin,kappa-tefluthrin, carbofuran, carbosulfan, oxamyl, thiodicarb,chlorpyrifos, chlorpyrifos-e, chlorpyrifos-methyl, diazinon, phorate,terbufos, fipronil, acetamiprid, clothianidin, imidacloprid,thiacloprid, thiamethoxam, abamectin, flonicamid, flubendiamide,bifenthrin, lambda-cyhalothrin, cypermethrin, zeta-cypermethrin,deltamethrin, or any mixtures thereof.

More preferred are mixtures of the Bacillus thuringiensis RTI545 strainwith clothianidin, thiamethoxam, imidacloprid, tefluthrin, fipronil,chlorpyrifos-e, tebupirimfos, bifenthrin, cypermethrin,zeta-cypermethrin, gamma-cyhalothrin, oxamyl, chlorantraniliprole,cyantraniliprole, cyclaniliprole, or mixtures thereof.

In one or more embodiments, the insecticide can comprise bifenthrin andthe composition can be formulated as a liquid. In one or moreembodiments, the insecticide can comprise bifenthrin and clothianidin.In one or more embodiments, the insecticide can comprise bifenthrin andclothianidin and the composition can be formulated as a liquid. In oneor more embodiments, the insecticide can comprise bifenthrin orzeta-cypermethrin. In one or more embodiments, the composition can beformulated as a liquid and the insecticide can comprise bifenthrin orzeta-cypermethrin.

In one embodiment, the chemical insecticide includes bifenthrin. In oneembodiment, the chemical insecticide includes bifenthrin and thecomposition further includes a hydrated aluminum-magnesium silicate, andat least one dispersant selected from the group consisting of a sucroseester, a lignosulfonate, an alkylpolyglycoside, a naphthalenesulfonicacid formaldehyde condensate and a phosphate ester. The bifenthrininsecticide can be present at a concentration ranging from 0.1 g/ml to0.2 g/ml. The bifenthrin insecticide can be present at a concentrationof 0.17 g/ml. The rate of application of the bifenthrin insecticide canbe in the range of from about 0.1 gram of bifenthrin per hectare (gai/ha) to about 1000 g ai/ha, more preferably in a range of from about 1g ai/ha to about 100 g ai/ha.

The bifenthrin can be preferably present in a concentration of from 1.0%by weight to 35% by weight, more particularly, from 15% by weight to 25%by weight based upon the total weight of all components in thecomposition. The bifenthrin insecticide composition can be formulated ina manner suitable for mixture as a liquid with a fertilizer.

Fungicides: B0) benzovindiflupyr, anitiperonosporics (such asametoctradin, amisulbrom, benthiavalicarb, cyazofamid, cymoxanil,dimethomorph, ethaboxam, famoxadone, fenamidone, flumetover, flumorph,fluopicolide, iprovalicarb, mandipropamid, valifenalate, benalaxyl,benalaxyl-M, furalaxyl, metalaxyl, and metalaxyl-M), ametoctradin,amisulbrom, copper salts (e.g., copper hydroxide, copper oxychloride,copper sulfate, copper persulfate), boscalid, thiflumazide, flutianil,furalaxyl, thiabendazole, benodanil, mepronil, isofetamid, fenfuram,bixafen, fluxapyroxad, penflufen, sedaxane, coumoxystrobin,enoxastrobin, flufenoxystrobin, pyraoxystrobin, pyrametostrobin,triclopyricarb, fenaminstrobin, metominostrobin, pyribencarb,meptyldinocap, fentin acetate, fentin chloride, fentin hydroxide,oxytetracycline, chlozolinate, chloroneb, tecnazene, etridiazole,iodocarb, prothiocarb, various Bacillus strains (e.g., strainsidentified as CH200, CH201, RTI184, RTI301, QST713, FZB24, MBI600,D747), extract from Melaleuca alternifolia, extract from Lupinus albusdoce, BLAD polypeptide, pyrisoxazole, oxpoconazole, etaconazole,fenpyrazamine, fenpicoxamide, mefentrifluconazole, naftifine,terbinafine, validamycin, pyrimorph, valifenalate, fthalide,probenazole, isotianil, laminarin, extract from Reynoutriasachalinensis, phosphorous acid and salts, teclofthalam, triazoxide,pyriofenone, organic oils, potassium bicarbonate, chlorothalonil,fluoroimide; B1) azoles, including bitertanol, bromuconazole,cyproconazole, difenoconazole, diniconazole, enilconazole,epoxiconazole, fluquinconazole, fenbuconazole, flusilazole, flutriafol,hexaconazole, imibenconazole, ipconazole, metconazole, myclobutanil,penconazole, propiconazole, prothioconazole, simeconazole, triadimefon,triadimenol, tebuconazole, tetraconazole, triticonazole, prochloraz,pefurazoate, imazalil, triflumizole, cyazofamid, benomyl, carbendazim,thiabendazole, fuberidazole, ethaboxam, etridiazole and hymexazole,azaconazole, diniconazole-M, oxpoconazol, paclobutrazol, uniconazol,1-(4-chloro-phenyl)-2-([1,2,4]triazol-1-yl)-cycloheptanol andimazalilsulfphate; B2) strobilurins, including azoxystrobin,dimoxystrobin, enestroburin, fluoxastrobin, kresoxim-methyl,methominostrobin, orysastrobin, picoxystrobin, pyraclostrobin,trifloxystrobin, enestroburin, methyl(2-chloro-5-[1-(3-methylbenzyloxyimino)ethyl]benzyl)carbamate, methyl(2-chloro-5-[1-(6-methylpyridin-2-ylmethoxyimino)ethyl]benzyl)carbamateand methyl2-(ortho-(2,5-dimethylphenyloxymethylene)-phenyl)-3-methoxyacrylate,2-(2-(6-(3-chloro-2-methyl-phenoxy)-5-fluoro-pyrimidin-4-yloxy)-phenyl)-2-methoxyimino-N-methyl-acetamideand3-methoxy-2-(2-(N-(4-methoxy-phenyl)-cyclopropanecarboximidoylsulfanylmethyl)-phenyl)-acrylicacid methyl ester; B3) carboxamides, including carboxin, benalaxyl,benalaxyl-M, fenhexamid, flutolanil, furametpyr, mepronil, metalaxyl,mefenoxam, ofurace, oxadixyl, oxycarboxin, penthiopyrad, isopyrazam,thifluzamide, tiadinil,3,4-dichloro-N-(2-cyanophenyl)isothiazole-5-carboxamide, dimethomorph,flumorph, flumetover, fluopicolide (picobenzamid), zoxamide,carpropamid, diclocymet, mandipropamid,N-(2-(4-[3-(4-chlorophenyl)prop-2-ynyloxy]-3-methoxyphenyl)ethyl)-2-methanesulfonyl-amino-3-methylbutyramide,N-(2-(4-[3-(4-chloro-phenyl)prop-2-ynyloxy]-3-methoxy-phenyl)ethyl)-2-ethanesulfonylamino-3-methylbutyramide,methyl3-(4-chlorophenyl)-3-(2-isopropoxycarbonylamino-3-methyl-butyrylamino)propionate,N-(4′-bromobiphenyl-2-yl)-4-difluoromethyl-2-methylthiazole-5-carboxamide,N-(4′-trifluoromethyl-biphenyl-2-yl)-4-difluoromethyl-2-methylthiazole-5-carboxamide,N-(4′-chloro-3′-fluorobiphenyl-2-yl)-4-difluoromethyl-2-methylthiazole-5-carboxamide,N-(3′,4′-dichloro-4-fluorobiphenyl-2-yl)-3-difluoro-methyl-1-methylpyrazole-4-carboxamide,N-(3′,4′-dichloro-5-fluorobiphenyl-2-yl)-3-difluoromethyl-1-methylpyrazole-4-carboxamide,N-(2-cyano-phenyl)-3,4-dichloroisothiazole-5-carboxamide,2-amino-4-methylthiazole-5-carboxanilide,2-chloro-N-(1,1,3-trimethyl-indan-4-yl)-nicotinamide,N-(2-(1,3-dimethylbutyl)-phenyl)-1,3-dimethyl-5-fluoro-1H-pyrazole-4-carboxamide,N-(4′-chloro-3′,5-difluoro-biphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide,N-(4′-chloro-3′,5-difluoro-biphenyl-2-yl)-3-trifluoromethyl-1-methyl-1H-pyrazole-4-carboxamide,N-(3′,4′-dichloro-5-fluorobiphenyl-2-yl)-3-trifluoromethyl-1-methyl-1H-pyrazole-4-carboxamide,N-(3′,5-difluoro-4′-methyl-biphenyl-2-yl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide,N-(3′,5-difluoro-4′-methyl-biphenyl-2-yl)-3-trifluoromethyl-1-methyl-1H-pyrazole-4-carboxamide,N-(cis-2-bicyclopropyl-2-yl-phenyl)-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide,N-(trans-2-bicyclopropyl-2-yl-phenyl)-3-difluoro-methyl-1-methyl-1H-pyrazole-4-carboxamide,fluopyram,N-(3-ethyl-3,5-5-trimethyl-cyclohexyl)-3-formylamino-2-hydroxy-benzamide,oxytetracyclin, silthiofam, N-(6-methoxy-pyridin-3-yl)cyclopropanecarboxamide, 2-iodo-N-phenyl-benzamide,N-(2-bicyclopropyl-2-yl-phenyl)-3-difluormethyl-1-methylpyrazol-4-ylcarboxamide,N-(3′,4′,5′-trifluorobiphenyl-2-yl)-1,3-di methylpyrazol-4-ylcarboxamide,N-(3′,4′,5′-trifluorobiphenyl-2-yl)-1,3-dimethyl-5-fluoropyrazol-4-yl-carboxamide,N-(3′,4′,5′-trifluorobiphenyl-2-yl)-5-chloro-1,3-dimethyl-pyrazol-4-ylcarboxamide,N-(3′,4′,5′-trifluorobiphenyl-2-yl)-3-fluoromethyl-1-methylpyrazol-4-ylcarboxamide,N-(3′,4′,5′-trifluorobiphenyl-2-yl)-3-(chlorofluoromethyl)-1-methylpyrazol-4-ylcarboxamide,N-(3′,4′,5′-trifluorobiphenyl-2-yl)-3-difluoromethyl-1-methylpyrazol-4-ylcarboxamide,N-(3′,4′,5′-trifluorobiphenyl-2-yl)-3-difluoromethyl-5-fluoro-1-methylpyrazol-4-ylcarboxamide,N-(3′,4′,5′-trifluorobiphenyl-2-yl)-5-chloro-3-difluoromethyl-1-methylpyrazol-4-ylcarboxamide,N-(3′,4′,5′-trifluorobiphenyl-2-yl)-3-(chlorodifluoromethyl)-1-methylpyrazol-4-ylcarboxamide,N-(3′,4′,5′-trifluorobiphenyl-2-yl)-1-methyl-3-trifluoromethylpyrazol-4-ylcarboxamide,N-(3′,4′,5′-trifluorobiphenyl-2-yl)-5-fluoro-1-methyl-3-trifluoromethylpyrazol-4-ylcarboxamide,N-(3′,4′,5′-trifluorobiphenyl-2-yl)-5-chloro-1-methyl-3-trifiuoromethylpyrazol-4-ylcarboxamide,N-(2′,4′,5′-trifluorobiphenyl-2-yl)-1,3-dimethylpyrazol-4-ylcarboxamide,N-(2′,4′,5′-trifluorobiphenyl-2-yl)-1,3-dimethyl-5-fluoropyrazol-4-ylcarboxamide,N-(2′,4′,5′-trifluorobiphenyl-2-yl)-5-chloro-1,3-dimethylpyrazol-4-ylcarboxamide,N-(2′,4′,5′-trifluorobiphenyl-2-yl)-3-fluoromethyl-1-methylpyrazol-4-ylcarboxamide,N-(2′,4′,5′-trifluorobiphenyl-2-yl)-3-(chlorofluoromethyl)-1-methylpyrazol-4-ylcarboxamide,N-(2′,4′,5′-trifluorobiphenyl-2-yl)-3-difluoromethyl-1-methylpyrazol-4-ylcarboxamide,N-(2′,4′,5′-trifluorobiphenyl-2-yl)-3-difluoromethyl-5-fluoro-1-methylpyrazol-4-ylcarboxamide,N-(2′,4′,5′-trifluorobiphenyl-2-yl)-5-chloro-3-difluoromethyl-1-methylpyrazol-4-ylcarboxamide,N-(2′,4′,5′-trifluorobiphenyl-2-yl)-3-(chlorodifluoromethyl)-1-methylpyrazol-4-ylcarboxamide,N-(2′,4′,5′-trifluorobiphenyl-2-yl)-1-methyl-3-trifluoromethylpyrazol-4-ylcarboxamide,N-(2′,4′,5′-trifluorobiphenyl-2-yl)-5-fluoro-1-methyl-3-trifluoromethylpyrazol-4-ylcarboxamide,N-(2′,4′,5′-trifluorobiphenyl-2-yl)-5-chloro-1-methyl-3-trifluoromethylpyrazol-4-ylcarboxamide,N-(3′,4′-dichloro-3-fluorobiphenyl-2-yl)-1-methyl-3-trifluoromethyl-1H-pyrazole-4-carboxamide,N-(3′,4′-dichloro-3-fluorobiphenyl-2-yl)-1-methyl-3-difluoromethyl-1H-pyrazole-4-carboxamide,N-(3′,4′-difluoro-3-fluorobiphenyl-2-yl)-1-methyl-3-trifluoromethyl-1H-pyrazole-4-carboxamide,N-(3′,4′-difluoro-3-fluorobiphenyl-2-yl)-1-methyl-5-difluoromethyl-1H-pyrazole-4-carboxamide,N-(3′-chloro-4′-fluoro-3-fluorobiphenyl-2-yl)-1-methyl-3-difluoromethyl-1H-pyrazole-4-carboxamide,N-(3′,4′-dichloro-4-fluorobiphenyl-2-yl)-1-methyl-3-trifluoromethyl-1H-pyrazole-4-carboxamide,N-(3′,4′-difluoro-4-fluorobiphenyl-2-yl)-1-methyl-S-trifluoromethyl-1H-pyrazole-4-carboxamide,N-(3′,4′-dichloro-4-fluorobiphenyl-2-yl)-1-methyl-3-difluoromethyl-1H-pyrazole-4-carboxamide,N-(3′,4′-difluoro-4-fluorobiphenyl-2-yl)-1-methyl-3-difluoromethyl-1H-pyrazole-4-carboxamide,N-(3′-chloro-4′-fluoro-4-fluorobiphenyl-2-yl)-1-methyl-5-difluoromethyl-1H-pyrazole-4-carboxamide,N-(3′,4′-dichloro-5-fluorobiphenyl-2-yl)-1-methyl-3-trifluoromethyl-1H-pyrazole-4-carboxamide,N-(3′,4′-difluoro-5-fluorobiphenyl-2-yl)-1-methyl-3-trifluoromethyl-1H-pyrazole-4-carboxamide,N-(3′,4′-dichloro-5-fluorobiphenyl-2-yl)-1-methyl-S-difluoromethyl-1H-pyrazole-carboxamide,N-(3′,4′-difluoro-5-fluorobiphenyl-2-yl)-1-methyl-3-difluoromethyl-1H-pyrazole-4-carboxamide,N-(3′,4′-dichloro-5-fluorobiphenyl-2-yl)-1,3-dimethyl-1H-pyrazole-4-carboxamide,N-(3′-chloro-4′-fluoro-5-fluorobiphenyl-2-yl)-1-methyl-3-difluoromethyl-1H-pyrazole-4-carboxamide,N-(4′-fluoro-4-fluorobiphenyl-2-yl)-1-methyl-3-trifluoromethyl-1H-pyrazole-4-carboxamide,N-(4′-fluoro-5-fluorobiphenyl-2-yl)-1-methyl-3-trifluoromethyl-1H-pyrazole-4-carboxamide,N-(4′-chloro-5-fluorobiphenyl-2-yl)-1-methyl-3-trifluoromethyl-1H-pyrazole-4-carboxamide,N-(4′-methyl-5-fluorobiphenyl-2-yl)-1-methyl-3-trifluoromethyl-1H-pyrazole-4-carboxamide,N-(4′-fluoro-5-fluorobiphenyl-2-yl)-1,3-dimethyl-1H-pyrazole-4-carboxamide,N-(4′-chloro-5-fluorobiphenyl-2-yl)-1,3-dimethyl-1H-pyrazole-4-carboxamide,N-(4′-methyl-5-fluorobiphenyl-2-yl)-1,3-dimethyl-1H-pyrazole-4-carboxamide,N-(4′-fluoro-6-fluorobiphenyl-2-yl)-1-methyl-3-trifluoromethyl-1H-pyrazole-4-carboxamide,N-(4′-chloro-6-fluorobiphenyl-2-yl)-1-methyl-3-trifluoromethyl-1H-pyrazole-4-carboxamide,N-[2-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl]-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamide,N-[4′-(trifluoromethylthio)-biphenyl-2-yl]-3-difluoromethyl-1-methyl-1H-pyrazole-4-carboxamideandN-[4′-(trifluoromethylthio)-biphenyl-2-yl]-1-methyl-3-trifluoromethyl-1-methyl-1H-pyrazole-4-carboxamide,3-difluoromethyl-N-(7-fluoro-1,1,3-trimethyl-4-indanyl)-1-methyl-4-pyrazolecarboxamide(fluindapyr),4-difluoromethyl-N-(7-fluoro-1,1,3-trimethyl-4-indanyl)-2-methyl-5-thiazolecarboxamide,3-difluoromethyl-1-methyl-N-(1,1,3,7-tetramethyl-4-indanyl)-pyrazolecarboxamide,4-difluoromethyl-2-methyl-N-(1,1,3,7-tetramethyl-4-indanyl)-5-thiazolecarboxamide,3-difluoromethyl-1-methyl-N-(7-methoxy-1,1,3-trimethyl-4-indanyl)-4-pyrazolecarboxamide,4-difluoromethyl-2-methyl-N-(7-methoxy-1,1,3-trimethyl-4-indanyl)-5-thiazolecarboxamide,3-difluoromethyl-1-methyl-N-(7-methylthio-1,1,3-trimethyl-4-indanyl)-4-pyrazolecarboxamide,4-difluoromethyl-2-methyl-N-(7-methylthio-1,1,3-trimethyl-4-indanyl)-5-thiazolecarboxamide,3-difluoromethyl-1-methyl-N-(7-trifluoromethoxy-1,1,3-trimethyl-4-indanyl)-4-pyrazolecarboxamide,4-difluoromethyl-2-methyl-N-(7-trifluoromethoxy-1,1,3-trimethyl-4-indanyl)-5-thiazolecarboxamide,3-difluoromethyl-N-(7-fluoro-1,1,3-trimethyl-4-indanyl)-4-furazancarboxamide,4-difluoromethyl-N-(7-fluoro-1,1,3-trimethyl-4-indanyl)-2-methylthio-5-pyrimidinecarboxamide,3-difluoromethyl-N-(7-chloro-1,1,3-trimethyl-4-indanyl)-1-methyl-4-pyrazolecarboxamide,3-difluoromethyl-N-(7-chloro-1,1-diethyl-3-methyl-4-indanyl)-1-methyl-4-pyrazolecarboxamide,or4-difluoromethyl-N-(7-fluoro-1,1,3-trimethyl-4-indanyl)-5-thiadiazolecarboxamide;B4) heterocyclic compounds, including fluazinam, pyrifenox, bupirimate,cyprodinil, fenarimol, ferimzone, mepanipyrim, nuarimol, pyrimethanil,triforine, fenpiclonil, fludioxonil, aldimorph, dodemorph,fenpropimorph, tridemorph, fenpropidin, iprodione, procymidone,vinclozolin, famoxadone, fenamidone, octhilinone, probenazole,5-chloro-7-(4-methyl-piperidin-1-yl)-6-(2,4,6-trifluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine,anilazine, diclomezine, pyroquilon, proquinazid, tricyclazole,2-butoxy-6-iodo-3-propylchromen-4-one, acibenzolar-S-methyl, captafol,captan, dazomet, folpet, fenoxanil, quinoxyfen,N,N-dimethyl-3-(3-bromo-6-fluoro-2-methylindole-1-sulfonyl)-[1,2,4]triazole-1-sulfonamide,5-ethyl-6-octyl-[1,2,4]triazolo[1,5-a]pyrimidin-2,7-diamine,2,3,5,6-tetrachloro-4-methanesulfonyl-pyridine,3,4,5-trichloro-pyridine-2,6-di-carbonitrile,N-(1-(5-bromo-3-chloro-pyridin-2-yl)-ethyl)-2,4-dichloro-nicotinamide,N-((5-bromo-3-chloro pyridin-2-yl)-methyl)-2,4-dichloro-nicotinamide,diflumetorim, nitrapyrin, dodemorphacetate, fluoroimid, blasticidin-S,chinomethionat, debacarb, difenzoquat, difenzoquat-methylsulphat,oxolinic acid and piperalin; B5) carbamates, including mancozeb, maneb,metam, methasulphocarb, metiram, ferbam, propineb, thiram, zineb, ziram,diethofencarb, iprovalicarb, benthiavalicarb, propamocarb, propamocarbhydrochlorid, 4-fluorophenylN-(1-(1-(4-cyanophenyl)-ethanesulfonyl)but-2-yl)carbamate, methyl3-(4-chloro-phenyl)-3-(2-isopropoxycarbonylamino-3-methyl-butyrylamino)propanoate;or B6) other fungicides, including guanidine, dodine, dodine free base,iminoctadine, guazatine, antibiotics: kasugamycin, oxytetracyclin andits salts, streptomycin, polyoxin, validamycin A, nitrophenylderivatives: binapacryl, dinocap, dinobuton, sulfur-containingheterocyclyl compounds: dithianon, isoprothiolane, organometalliccompounds: fentin salts, organophosphorus compounds: edifenphos,iprobenfos, fosetyl, fosetyl-aluminum, phosphorous acid and its salts,pyrazophos, tolclofos-methyl, organochlorine compounds: dichlofluanid,flusulfamide, hexachloro-benzene, phthalide, pencycuron, quintozene,thiophanate, thiophanate-methyl, tolylfluanid, others: cyflufenamid,cymoxanil, dimethirimol, ethirimol, furalaxyl, metrafenone andspiroxamine, guazatine-acetate, iminoctadine-triacetate,iminoctadine-tris(albesilate), kasugamycin hydrochloride hydrate,dichlorophen, pentachlorophenol and its salts,N-(4-chloro-2-nitro-phenyl)-N-ethyl-4-methyl-benzenesulfonamide,dicloran, nitrothal-isopropyl, tecnazen, biphenyl, bronopol,diphenylamine, mildiomycin, oxincopper, prohexadione calcium,N-(cyclopropylmethoxyimino-(6-difluoromethoxy-2,3-difluoro-phenyl)-methyl)-2-phenylacetamide,N′-(4-(4-chloro-3-trifluoromethyl-phenoxy)-2,5-dimethyl-phenyl)-N-ethyl-N-methylformamidine,N′-(4-(4-fluoro-3-trifluoromethyl-phenoxy)-2,5-dimethyl-phenyl)-N-ethyl-N-methylformamidine,N′-(2-methyl-5-trifluormethyl-4-(3-trimethylsilanyl-propoxy)-phenyl)-N-ethyl-N-methylformamidineandN′-(5-difluormethyl-2-methyl-4-(3-trimethylsilanyl-propoxy)-phenyl)-N-ethyl-N-methylformamidine.

Of note are mixtures of the Bacillus thuringiensis RTI545 strain withother biocontrol strains including Bacillus subtilis strains such asCH201 or QST713 or MBI600 or RTI477, Bacillus licheniformis strains suchas CH200 or RTI184, Bacillus velezensis strains such as RTI301, Bacillussubtilis var. amyloliquefaciens FZB24, or Bacillus amyloliquefaciensD747, or combinations thereof for fungal disease control.

In the compositions and methods of the present disclosure, thecomposition can include a chemical fungicide.

Preferred are mixtures of the Bacillus thuringiensis RTI545 strain withchemical fungal control agents comprising thiabendazole, fluxapyroxad,penflufen, sedaxane, bitertanol, cyproconazole, difenoconazole,fluquinconazole, flutriafol, ipconazole, myclobutanil, prothioconazole,triadimefon, triadimenol, tebuconazole, triticonazole, prochloraz,imazalil, benomyl, carbendazim, hymexazole, azoxystrobin, fluoxastrobin,pyraclostrobin, trifloxystrobin, carboxin, flutolanil, metalaxyl,mefenoxam, penthiopyrad, fluopyram, silthiofam, fluazinam, pyrimethanil,fludioxonil, iprodione, tricyclazole, captan, dazomet, mancozeb, metam,thiram, guazatine, tolclofos-methyl, pencycuron, thiophanate-methyl,fenpicoxamide, mefentrifluconazole, fluindapyr, or any mixtures thereof.

More preferred are mixtures of the Bacillus thuringiensis RTI545 strainwith fludioxonil, prothioconazole, mefenoxam, metalaxyl, tebuconazole,difenoconazole, thiram, carboxin, carbendazim, triticonazole,pencycuron, imazalil, pyraclostrobin, sedaxane, trifloxystrobin,fluquinconazole, fluoxastrobin, azoxystrobin, flutriafol, fluxapyroxad,penthiopyrad, fenpicoxamide, mefentrifluconazole, fluindapyr or mixturesthereof.

Herbicides: C1) acetyl-CoA carboxylase inhibitors (ACC), for examplecyclohexenone oxime ethers, such as alloxydim, clethodim, cloproxydim,cycloxydim, sethoxydim, tralkoxydim, butroxydim, clefoxydim ortepraloxydim; phenoxyphenoxypropionic esters, such asclodinafop-propargyl, cyhalofop-butyl, diclofop-methyl,fenoxaprop-ethyl, fenoxaprop-P-ethyl, fenthiapropethyl, fluazifop-butyl,fluazifop-P-butyl, haloxyfop-ethoxyethyl, haloxyfop-methyl,haloxyfop-P-methyl, isoxapyrifop, propaquizafop, quizalofop-ethyl,quizalofop-P-ethyl or quizalofop-tefuryl; or arylaminopropionic acids,such as flamprop-methyl or flamprop-isopropyl; C2 acetolactate synthaseinhibitors (ALS), for example imidazolinones, such as imazapyr,imazaquin, imazamethabenz-methyl (imazame), imazamox, imazapic orimazethapyr; pyrimidyl ethers, such as pyrithiobac-acid,pyrithiobac-sodium, bispyribac-sodium. KIH-6127 or pyribenzoxym;sulfonamides, such as florasulam, flumetsulam or metosulam; orsulfonylureas, such as amidosulfuron, azimsulfuron, bensulfuron-methyl,chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron,ethametsulfuron-methyl, ethoxysulfuron, flazasulfuron,halosulfuron-methyl, imazosulfuron, metsulfuron-methyl, nicosulfuron,primisulfuron-methyl, prosulfuron, pyrazosulfuron-ethyl, rimsulfuron,sulfometuron-methyl, thifensulfuron-methyl, triasulfuron,tribenuron-methyl, triflusulfuron-methyl, tritosulfuron, sulfosulfuron,foramsulfuron or iodosulfuron; C3) amides, for example allidochlor(CDAA), benzoylprop-ethyl, bromobutide, chiorthiamid, diphenamid,etobenzanidibenzchlomet), fluthiamide, fosamin or monalide; C4) auxinherbicides, for example pyridinecarboxylic acids, such as clopyralid orpicloram; or 2,4-D or benazolin; C5) auxin transport inhibitors, forexample naptalame or diflufenzopyr; C6) carotenoid biosynthesisinhibitors, for example benzofenap, clomazone (dimethazone),diflufenican, fluorochloridone, fluridone, pyrazolynate, pyrazoxyfen,isoxaflutole, isoxachlortole, mesotrione, sulcotrione (chlormesulone),ketospiradox, flurtamone, norflurazon or amitrol; C7)enolpyruvylshikimate-3-phosphate synthase inhibitors (EPSPS), forexample glyphosate or sulfosate; C8) glutamine synthetase inhibitors,for example bilanafos (bialaphos) or glufosinate-ammonium; C9) lipidbiosynthesis inhibitors, for example anilides, such as anilofos ormefenacet; chloroacetanilides, such as dimethenamid, S-dimethenamid,acetochlor, alachlor, butachlor, butenachlor, diethatyl-ethyl,dimethachlor, metazachlor, metolachlor, S-metolachlor, pretilachlor,propachlor, prynachlor, terbuchlor, thenylchlor or xylachlor; thioureas,such as butylate, cycloate, di-allate, dimepiperate, EPTC. esprocarb,molinate, pebulate, prosulfocarb, thiobencarb (benthiocarb), tri-allateor vemolate; or benfuresate or perfluidone; C10) mitosis inhibitors, forexample carbamates, such as asulam, carbetamid, chlorpropham, orbencarb,pronamid (propyzamid), propham or tiocarbazil; dinitroanilines, such asbenefin, butralin, dinitramin, ethalfluralin, fluchloralin, oryzalin,pendimethalin, prodiamine or trifluralin; pyridines, such as dithiopyror thiazopyr; or butamifos, chlorthal-dimethyl (DCPA) or maleichydrazide; C11) protoporphyrinogen IX oxidase inhibitors, for examplediphenyl ethers, such as acifluorfen, acifluorfen-sodium, aclonifen,bifenox, chlomitrofen (CNP), ethoxyfen, fluorodifen,fluoroglycofen-ethyl, fomesafen, furyloxyfen, lactofen, nitrofen,nitrofluorfen or oxyfluorfen; oxadiazoles, such as oxadiargyl oroxadiazon; cyclic imides, such as azafenidin, butafenacil,carfentrazone-ethyl, cinidon-ethyl, flumiclorac-pentyl, flumioxazin,flumipropyn, flupropacil, fluthiacet-methyl, sulfentrazone orthidiazimin; or pyrazoles, such as ET-751.JV 485 or nipyraclofen; C12)photosynthesis inhibitors, for example propanil, pyridate or pyridafol;benzothiadiazinones, such as bentazone; dinitrophenols, for examplebromofenoxim, dinoseb, dinoseb-acetate, dinoterb or DNOC; dipyridylenes,such as cyperquat-chloride, difenzoquat-methylsulfate, diquat orparaquat-dichloride; ureas, such as chlorbromuron, chlorotoluron,difenoxuron, dimefuron, diuron, ethidimuron, fenuron, fluometuron,isoproturon, isouron, linuron, methabenzthiazuron, methazole,metobenzuron, metoxuron, monolinuron, neburon, siduron or tebuthiuron;phenols, such as bromoxynil or ioxynil; chloridazon; triazines, such asametryn, atrazine, cyanazine, desmein, dimethamethryn, hexazinone,prometon, prometryn, propazine, simazine, simetryn, terbumeton,terbutryn, terbutylazine or trietazine; triazinones, such as metamitronor metribuzin; uracils, such as bromacil, lenacil or terbacil; orbiscarbamates, such as desmedipham or phenmedipham; C13) synergists, forexample oxiranes, such as tridiphane; C14) CIS cell wall synthesisinhibitors, for example isoxaben or dichlobenil; C15) various otherherbicides, for example dichloropropionic acids, such as dalapon;dihydrobenzofurans, such as ethofumesate; phenylacetic acids, such aschlorfenac (fenac); or aziprotryn, barban, bensulide, benzthiazuron,benzofluor, buminafos, buthidazole, buturon, cafenstrole, chlorbufam,chlorfenprop-methyl, chloroxuron, cinmethylin, cumyluron, cycluron,cyprazine, cyprazole, dibenzyluron, dipropetryn, dymron,eglinazin-ethyl, endothall, ethiozin, flucabazone, fluorbentranil,flupoxam, isocarbamid, isopropalin, karbutilate, mefluidide, monuron,napropamide, napropamide-M, napropanilide, nitralin, oxaciclomefone,phenisopham, piperophos, procyazine, profluralin, pyributicarb,secbumeton, sulfallate (CDEC), terbucarb, triaziflam, triazofenamid ortrimeturon; or their environmentally compatible salts.

Nematicides or bionematicides: benomyl, cloethocarb, aldoxycarb,tirpate, diamidafos, fenamiphos, cadusafos, dichlofenthion, ethoprophos,fensulfothion, fosthiazate, heterophos, isamidofof, isazofos,phosphocarb, thionazin, imicyafos, mecarphon, acetoprole, benclothiaz,oxamyl, chloropicrin, dazomet, fluensulfone, 1,3-dichloropropene(telone), dimethyl disulfide, metam sodium, metam potassium, metam salt(all MITC generators), methyl bromide, biological soil amendments (e.g.,mustard seeds, mustard seed extracts), steam fumigation of soil, allylisothiocyanate (AITC), dimethyl sulfate, furfural (aldehyde),fluazaindolizine (DPX-Q8U80), fluopyram, or tioxazafen.

Preferred are mixtures of the Bacillus thuringiensis RTI545 strain withchemical nematode control agents comprising benomyl, fenamiphos,cadusafos, ethoprophos, fosthiazate, chloropicrin, dazomet,fluensulfone, oxamyl, 1,3-dichloropropene (telone), metam sodium, metampotassium, metam salt (all MITC generators), methyl bromide, allylisothiocyanate (AITC), fluazaindolizine (DPX-Q8U80), tioxazafen,fluopyram, or any mixtures thereof.

More preferred are mixtures of the Bacillus thuringiensis RTI545 strainwith cadusafos, ethoprophos, fosthiazate, fluensulfone, oxamyl,fluazaindolizine (DPX-Q8U80), tioxazafen, or any mixtures thereof. Inthe compositions and methods of the present disclosure, the compositioncan include the nematicide cadusafos.

Suitable plant growth regulators of the present invention include thefollowing: Plant Growth Regulators: D1) Antiauxins, such as clofibricacid, 2,3,5-tri-iodobenzoic acid; D2) Auxins such as 4-CPA, 2,4-D,2,4-DB, 2,4-DEP, dichlorprop, fenoprop, IAA, IBA, naphthaleneacetamide,α-naphthaleneacetic acids, 1-naphthol, naphthoxyacetic acids, potassiumnaphthenate, sodium naphthenate, 2,4,5-T; D3) cytokinins, such as 2iP,benzyladenine, 4-hydroxyphenethyl alcohol, kinetin, zeatin; D4)defoliants, such as calcium cyanamide, dimethipin, endothal, ethephon,merphos, metoxuron, pentachlorophenol, thidiazuron, tribufos; D5)ethylene inhibitors, such as aviglycine, 1-methylcyclopropene; D6)ethylene releasers, such as ACC, etacelasil, ethephon, glyoxime; D7)gametocides, such as fenridazon, maleic hydrazide; D8) gibberellins,such as gibberellins, gibberellic acid; D9) growth inhibitors, such asabscisic acid, ancymidol, butralin, carbaryl, chlorphonium,chlorpropham, dikegulac, flumetralin, fluoridamid, fosamine, glyphosine,isopyrimol, jasmonic acid, maleic hydrazide, mepiquat, piproctanyl,prohydrojasmon, propham, tiaojiean, 2,3,5-tri-iodobenzoic acid; D10)morphactins, such as chlorfluren, chlorflurenol, dichlorflurenol,flurenol; D11) growth retardants, such as chlormequat, daminozide,flurprimidol, mefluidide, paclobutrazol, tetcyclacis, uniconazole; D12)growth stimulators, such as brassinolide, brassinolide-ethyl, DCPTA,forchlorfenuron, hymexazol, prosuler, triacontanol; D13) unclassifiedplant growth regulators, such as bachmedesh, benzofluor, buminafos,carvone, choline chloride, ciobutide, clofencet, cyanamide, cyclanilide,cycloheximide, cyprosulfamide, epocholeone, ethychlozate, ethylene,fuphenthiourea, furalane, heptopargil, holosulf, inabenfide, karetazan,lead arsenate, methasulfocarb, prohexadione, pydanon, sintofen,triapenthenol, trinexapac.

Chemical formulations of the present invention can be in any appropriateconventional form, for example an emulsion concentrate (EC), asuspension concentrate (SC), a suspo-emulsion (SE), a capsule suspension(CS), a water dispersible granule (WG), an emulsifiable granule (EG), awater in oil emulsion (EO), an oil in water emulsion (EW), amicro-emulsion (ME), an oil dispersion (OD), an oil miscible flowable(OF), an oil miscible liquid (OL), a soluble concentrate (SL), anexpandable foam (EF) suspension, an ultra-low volume suspension (SU), anultra-low volume liquid (UL), a dispersible concentrate (DC), a wettablepowder (WP), granules (G) of various sizes that, in embodiments, may bedeposited at the time of planting, or any technically feasibleformulation in combination with agriculturally acceptable adjuvants.

In embodiments, the composition may comprise: 0.5-99 weight % of abiologically pure culture of Bacillus thuringiensis RTI545 deposited asATCC No. PTA-122161, or a mutant thereof having all the identifyingcharacteristics thereof, of at not less than about 1×10¹¹ CFU/g, and anagriculturally acceptable adjuvant. In at least one embodiment, theactive ingredient comprising the Bacillus species is present in totalconcentrations ranging between 0.5% to about 95 weight % of theagricultural composition, such as wherein the Bacillus species ispresent in an amount independently selected from a lower limit of 1, 2,3, 4 or 5, 7, 8, or 10 weight % to an upper limit of 10, 15, 20, 25, 40,50, 60, 70, 80 or 90 weight % of the total composition. In anotherembodiment, agriculturally acceptable adjuvants constitute about 1% toabout 99.5%, such as from a lower limit of 1, 2, 3, 4 or 5 weight % toan upper limit of 10, 15, 20, 25, 40, 50, 60, 70, 80, 90, or 95 weight %of the total composition.

In embodiments, the adjuvant may be selected from the group consistingof liquid carriers, solid carriers, surface acting agents (surfactants),viscosity modifiers, thickeners, rheology additives, structuring agents,preservatives, biocides or biostatic agents, antifreezes,crystallization inhibitors, suspending agents, dyes, anti-oxidants,foaming agents, light absorbers, mixing auxiliaries, antifoams,complexing agents, neutralizing or pH-modifying substances and buffers,corrosion inhibitors, fragrances, wetting agents, take-up enhancers,micronutrients, plasticizers, glidants, lubricants, and dispersants.

Carriers can be liquid or solid. Adjuvants that may be used in suchformulations include surface active agents, viscosity modifiers such asthickeners, preservatives, biocides or biostatic agents, antifreezes,crystallization inhibitors, suspending agents, dyes, anti-oxidants,foaming agents, light absorbers, mixing auxiliaries, antifoams,complexing agents, neutralizing or pH-modifying substances and buffers,corrosion inhibitors, fragrances, wetting agents, take-up enhancers,micronutrients, plasticizers, glidants, lubricants, dispersants, andalso liquid and solid fertilizers.

In embodiments, the compositions of this invention may be formulated asa suspension concentrate (SC), wettable powder (WP) or wettable granule(WG). Other formulations types include water dispersible powders forslurry treatment (WS), oil dispersions (OD), granules for broadcastapplications (GR), capsule suspensions (CS), emulsifiable concentrates(EC), emulsions in water (EW), soluble concentrates (SL), mixedformulations of a CS and an SC (ZC), mixed formulations of an SC and anEW as suspo-emulsions (SE), an expandable foam (EF) suspension, or mixedformulations of a CS and an EW (ZW). In some embodiments thecompositions may be formulated as dusts, or powders, or granules thatcan be applied to the plant, plant part, seed, or soil as a dryformulation (e.g. a dry seed coating on peanuts or a dust, powder orgranules of various sizes for incorporation into soil).

Liquid carriers include solvents and co-solvents including water,petroleum ether, vegetable oils, acid anhydrides, amyl acetate, butylenecarbonate, cyclohexane, cyclohexanol, diacetone alcohol,1,2-dichloropropane, diethanolamine, diethylene glycol, diethyleneglycol abietate, diethylene glycol butyl ether, diethylene glycol ethylether, diethylene glycol methyl ether, 1,4-dioxane, dipropylene glycol,dipropylene glycol methyl ether, dipropylene glycol dibenzoate,diproxitol, alkylpyrrolidone, 2-ethylhexanol, ethylene carbonate,1,1,1-trichloroethane, alpha-pinene, d-limonene, ethyl lactate, ethyleneglycol, ethylene glycol butyl ether, ethylene glycol methyl ether,gamma-butyrolactone, glycerol, glycerol acetate, glycerol diacetate,glycerol triacetate, hexadecane, hexylene glycol, isobornyl acetate,isooctane, isophorone, isopropyl myristate, lactic acid, laurylamine,mesityl oxide, methoxypropanol, methyl laurate, methyl octanoate, methyloleate, methylene chloride, n-hexane, n-octylamine, octadecanoic acid,octylamine acetate, oleic acid, oleylamine, polyethylene glycol (PEG),propionic acid, propyl lactate, propylene carbonate, propylene glycol,propylene glycol methyl ether, triethyl phosphate, triethylene glycol,xylenesulfonic acid, paraffin, mineral oil, trichloroethylene,perchloroethylene, alcohols of higher molecular weight, such as amylalcohol, tetrahydrofurfuryl alcohol, hexanol, octanol, liquid amidessuch as N,N-dimethyloctanamide, N,N-dimethyldecanamide,N-methyl-N-(2-propylheptyl)-acetamide,N-methyl-N-(2-propylheptyl)-formamide, N-methyl-2-pyrrolidone and thelike. Preferably, liquid carriers are such that the biological activeagents remain essentially unchanged in the composition until after it isapplied to the locus of control. Water is generally the carrier ofchoice for diluting the concentrated formulations.

Suitable solid carriers include, for example, carbohydrates includingmono or di carbohydrates such as sucrose, oligo or poly-saccharides suchas maltodextrin or pectin, talc, titanium dioxide, pyrophyllite clay,attapulgite clay, kieselguhr, silica (silicon dioxide), limestone,bentonite, calcium montmorillonite water soluble salts such as sodium,potassium, magnesium, calcium or ammonium salts of acetate, carbonate,chloride, citrate, phosphate, or sulfate such as calcium carbonate,cottonseed husks, wheat flour, soybean flour, pumice, wood flour, groundwalnut shells, lignin and similar substances, yeast extracts, fish meal,or mixtures thereof. Notable solid carriers include maltodextrin,silica, calcium carbonate, or any mixtures thereof.

Surface active agents including surfactants, dispersants andemulsifiers, viscosity enhancing agents, solvents and other adjuvantsindependently may constitute between about 0.1% to about 25% of thefinal formulation by weight.

The compositions may contain a surface-active substance (surfactants,dispersants and emulsifiers) from a very large variety of substancesknown in the art that are also commercially available. Surface-activesubstances (described herein generally as surfactants) may be anionic,cationic, non-ionic or polymeric and they can be used as surfactants,dispersants, emulsifiers, wetting agents or suspending agents or forother purposes.

Surfactants belong to different classes such as cationic surfactants,anionic surfactants, non-ionic surfactants, ionic surfactants, andamphoteric surfactants. According to the invention, the surfactant canbe any surfactant or combination of two or more surfactants useful todisperse the biological active ingredients in the formulation or tankmix for application. The amounts of the surfactant in the compositionsof this invention may range from about 1 to about 15%, or about 1 toabout 10%, preferably about 3 to about 8%, and more preferably about 5to about 7% w/w.

Examples of some preferred surfactants include cationic, non-ionic,anionic and/or amphoteric surfactants.

Non-ionic surfactants suitable for this invention include ethoxylatedlinear alcohols, ethoxylated alkyl phenol, alkyl EO/PO copolymer,polyalkylene glycol monobutyl ether ethoxylated fatty acids/oils,sorbitan laurate, polysorbate, sorbitan oleate, ethoxylated fatty acidalcohols, or alkyl phenols, alkanolamides or alkyloamides (such asdiethanolamide, lauric acid monoisopropanolamide, and ethoxylatedmyristamide), xyethylene fatty acid esters, polyoxyethylene fattyalcohol ethers (such as alkylaryl polyglycol ethers),alkylphenol/alkylene oxide addition products, such as nonylphenolethoxylate; alcohol/alkylene oxide addition products, such astridecylalcohol ethoxylate.

Anionic surfactants include alkyl-, alkylaryl- and arylsulfonates orsalts thereof (such as sodium, potassium or calcium salts of laurylsarcosinate, alkylbenzenesulfonate, dodecylbenzenesulfonate,alkylnaphthalenesulfonates such as dibutylnaphthalenesulfonate, orC₁₄₋₁₆ olefin sulfonates), alkyl-, alkylaryl- and arylsulfates or saltsthereof (such as sodium, potassium or calcium salts of tridedethsulfate, lauryl sulfate, decyl sulfate, and diethanolammonium laurylsulfate) protein hydrolysates, derivatives of polycarboxylic acid (suchas ammonium lauryl ether carboxylate), olefin sulfonates (such as sodiumalpha olefin sulfonate), sarcosinates (such as ammonium cyclohexylpalmitoyl taurinate), succinates (such as disodium N-octadecylsulfosuccinamate), phosphorus derivatives (such as phosphoric acidesters and their equivalent salts).

Cationic surfactants include alkylbenzyltrimethylammonium chloride,ammonium lauryl sulfate and lauramine oxide.

In some embodiments, surfactants may be used as foaming agents allowingthe formulation to be foamable for applying to the seed or in-furrow atthe time of planting. The foamable composition can be optionally dilutedwith water and mixed with a pressurized gas such as air in a foamingchamber comprising a foaming medium such as a plurality of glass beads.

Suitable foaming agents may be nonionic surfactants includingalkanolamides or alkyloamides (such as cocamide diethanolamide, lauricacid monoisopropanolamide, and ethoxylated myristamide), xyethylenefatty acid esters, polyoxyethylene fatty alcohol ethers (such asalkylaryl polyglycol ethers) and fluorocarbons (such as ethoxylatedpolyfluorinated alcohol); anionic surfactants including alkyl-,alkylaryl- and arylsulfonates (such as sodium lauryl sarcosinate andsuch as sodium alkylbenzenesulfonate), alkyl-, alkylaryl- andarylsulfates, protein hydrolysates, derivatives of polycarboxylic acid(such as ammonium lauryl ether carboxylate), olefin sulfonates (such assodium alpha olefin sulfonate), sarcosinates (such as ammoniumcyclohexyl palinitoyl taurinate), succinates (such as disodiumN-octadecyl sulfosuccinamate), phosphorus derivatives (such asphosphoric acid esters and their equivalent salts); cationic surfactantsincluding alkylbenzyltrimethylammonium chloride; and amphotericsurfactants including betaine. Particularly preferred foaming agentsinclude sodium dodecylbenzene sulfonate (ex. Bio-Soft® D-40), sodiumC₁₄₋₁₆ olefin sulfonate (ex. Bioterge® AS-40), lauramine oxide (ex.Ammonyx® DO, Ammonyx® LO), ammonium lauryl sulfate (ex. Steol®), sodium(Cedepal® TD-407) and alkyl sulfates (ex Polystep® B-25). The totalconcentration of foaming agents in the formulation will be dependent onthe foaming agents used and may comprise between about 0.1% and about50% of the concentrated foamable formulation, preferably between about0.3% and about 30% more preferably between about 5% and 25% and evenmore preferably between about 17% and about 23%.

Notable embodiments include those wherein the volume of the foamgenerated by the formulation is reduced by 25% (or less) after about 45minutes or greater. Other surface active substances include soaps, suchas sodium stearate; dialkyl esters of sulfosuccinate salts, such assodium di(2-ethylhexyl)sulfosuccinate; sorbitol esters, such as sorbitololeate; quaternary amines, such as lauryltrimethylammonium chloride,polyethylene glycol esters of fatty acids, such as polyethylene glycolstearate; block copolymers of ethylene oxide and propylene oxide; andsalts of mono- and di-alkylphosphate esters.

Also suitable are silicone surfactants, especiallypolyalkyl-oxide-modified heptamethyltriloxanes which are commerciallyavailable e.g. as Silwet L-77®, and also perfluorinated surfactants.

Of these, some even more specific types of preferred surfactants includenon-ionic linear or branched alcohol ethoxylate surfactants, anionicphosphoric acid ester surfactants (sometimes referred to as “phosphateester” surfactants), and cationic ethoxylated tallow amine surfactants.

Notable surfactants (dispersants) comprise at least one alkylalkylpolyglycoside, preferably comprising C₈-C₁₄ alkyl groups. TheAgnique® products of BASF Corporation (Cognis) are representative. Inone embodiment, the alkyl d-glycopyranoside surfactant includes amixture of C₈-C₁₀ alkyl d-glucopyranosides, such as Agnique® PG8105-G.In another embodiment, the alkyl d-glucopyranoside surfactant includes amixture of C₉-C₁₁ alkyl d-glucopyranosides. A preferred product isAgnique® PG9116 which is a mixture of C₉-C₁₁ alkyl d-glucopyranosides,having a degree of polymerization of about 1.6 and ahydrophilic-lipophilic balance (HLB) of about 13.1.

Phosphate ester surfactants (dispersants) may comprise phosphate estersof alcohols, ethoxylated alcohols or ethoxylated phenol. They can be inthe free acid form or neutralized as the sodium, potassium or ammoniumsalts. The Dextrol® products of Ashland Corporation are representative,such as Dextrol® OC-180. The phosphate ester is preferably selected froma nonyl phenol phosphate ester and a tridecyl alcohol ethoxylatedphosphate potassium salt.

In another aspect, the composition may contain a thickener, viscositymodifier, rheology additive or structuring agent that stabilizeformulations such as suspension concentrates or oil dispersions againstsettling or sedimentation. Suitable thickeners are rice, starch, gumarabic, gum tragacanth, guar flour, British gum, starch ethers andstarch esters, gum resins, galactomannans, magnesium aluminum silicate,xanthan gum, carrageenan, cellulose derivatives, methyl cellulose,carboxymethylcellulose, alginates and combinations thereof. Other knowncommercial products may include Lattice NTC 50, Lattice NTC 60,methocel, clay, and veegum silica.

In another embodiment, the compositions of this invention may contain anantifreeze agent such as glycerine, ethylene glycol, propylene glycol,urea, calcium chloride, sodium nitrate, magnesium chloride and ammoniumsulfate.

Suitable preservatives include but are not limited to C₁₂ to C₁₅ alkylbenzoates, alkyl p-hydroxybenzoates, aloe vera extract, ascorbic acid,benzalkonium chloride, benzoic acid, benzoic acid esters of C₉ to C₁₅alcohols, butylated hydroxytoluene, butylated hydroxyanisole,tert-butylhydroquinone, castor oil, cetyl alcohols, chlorocresol, citricacid, cocoa butter, coconut oil, diazolidinyl urea, diisopropyl adipate,dimethyl polysiloxane, DMDM hydantoin, ethanol,ethylenediaminetetraacetic acid, fatty acids, fatty alcohols, hexadecylalcohol, hydroxybenzoate esters, iodopropynyl butylcarbamate, isononyliso-nonanoate, jojoba oil, lanolin oil, mineral oil, oleic acid, oliveoil, parabens, polyethers, polyoxypropylene butyl ether,polyoxypropylene cetyl ether, potassium sorbate, propyl gallate,silicone oils, sodium propionate, sodium benzoate, sodium bisulfite,sorbic acid, stearic fatty acid, sulfur dioxide, vitamin E, vitamin Eacetate and derivatives, esters, salts and mixtures thereof. Preferredpreservatives include sodium o-phenylphenate,5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one,and 1,2-benisothiazolin-3-one.

Antifoam agents such as Xiameter AFE-100, Dow Corning AFs, Dow Corning1520, 1530, or 1540 may also be used in the presently claimedformulations.

In embodiments, the composition may be a liquid suspension concentratecomprising water and at least one surface active agent, and one or moreadditional adjuvants. In embodiments, the one or more adjuvants may beselected from thickeners, viscosity modifiers, structuring agents orrheology additives, solvents, preservatives, antifreeze agents, andantifoam agents. Normally, the suspension concentrate is further dilutedwith water before delivery of the composition. In embodiments, theliquid composition may be a suspension concentrate comprising from 0.5to 20 weight % of a biologically pure culture of Bacillus thuringiensisRTI545, or a mutant thereof having all the identifying characteristicsthereof; 1 to 5 weight % of one or more surface active agent; and atleast one thickener, solvent, preservative, antifreeze agent, orantifoam agent each independently comprising up to about 1 weight % ofthe composition.

In other embodiments, the composition may be a liquid oil dispersioncomprising the solid active ingredients dispersed in oil such as avegetable oil and at least one surface active agent, and one or moreadditional adjuvants. In embodiments, the one or more adjuvants may beselected from thickeners, viscosity modifiers, structuring agents orrheology additives, solvents, preservatives, antifreeze agents, antifoamagents and the like. Normally, the oil dispersion is further dilutedwith water before delivery of the composition. In embodiments, theliquid composition may be a an oil dispersion comprising from 0.5 to 20weight % of a biologically pure culture of Bacillus thuringiensisRTI545, or a mutant thereof having all the identifying characteristicsthereof; 1 to 10 weight % of one or more surface active agent; and atleast one thickener, viscosity modifier, structuring agent, rheologyadditive, solvent, preservative, antifreeze agent, or antifoam agenteach independently comprising up to about 5 weight % of the composition.

In embodiments, the composition can be in the form of a dust, powder,granule, a dry wettable powder, a spreadable granule, or a dry wettablegranule and the biologically pure culture of Bacillus thuringiensisRTI545, or a mutant thereof having all the identifying characteristicsthereof can be present in an amount of from about 1.0×10⁸ CFU/g to about5×10¹³ CFU/g. In embodiments, the composition may comprise a solidcarrier selected from the group consisting of mono- or di-saccharides,oligo- or poly-saccharides, talc, titanium dioxide, pyrophyllite clay,attapulgite clay, kieselguhr, silica, limestone, bentonite, calciummontmorillonite, sodium, potassium, magnesium, calcium or ammonium saltsof acetate, carbonate, chloride, citrate, phosphate, or sulfate,cottonseed husks, wheat flour, soybean flour, pumice, wood flour, groundnut (such as peanut or walnut) shells, lignin, yeast extracts, fishmeal, or mixtures thereof.

In an embodiment, the composition comprises: 5-40% of a biologicallypure culture of not less than about 1×10¹¹ CFU/g; and maltodextrin,silica, calcium carbonate, or any mixtures thereof. In embodiments, thecomposition comprises 5-15% of maltodextrin.

In embodiments, the composition may comprise by weight %: 5-40% of abiologically pure culture of not less than about 1×10¹¹ CFU/g Bacillusthuringiensis RTI545, or a mutant thereof having all the identifyingcharacteristics thereof; 5-15% maltodextrin; 35-45% calcium carbonate;and 5-15% silica. In embodiments, the composition may be a wettablepowder formulation.

In an embodiment, the composition may be a wettable powder formulationcomprising by weight %: about 40% of a biologically pure culture of notless than about 1×10¹¹ CFU/g Bacillus thuringiensis RTI545, or a mutantthereof having all the identifying characteristics thereof; 10%maltodextrin; 40% calcium carbonate; and 10% silica.

In embodiments, the composition is useful in either plant seed treatmentor in-furrow applications for conferring protection against orcontrolling plant fungal pathogenic infection. For seed treatment, asolution or suspension of the composition can be applied to seed usingstandard seed treatment procedures. The composition may be applied tountreated seeds or seeds that have been treated with at least oneadditional crop protection agent as described herein. Alternatively, thecomposition may also be mixed with an additional crop protection agentfor seed treatment or in-furrow applications. In some embodiments, thecomposition may be applied to the foliage of the plant to be protected,optionally mixed with an additional crop protection agent.

In some embodiments of compositions and methods, the composition furtherincludes one or a combination of additional agricultural agent(s) suchas an insecticide, fungicide, nematicide, bacteriocide, herbicide, plantextract, plant growth regulator, or fertilizer as described hereinpresent in an amount suitable to benefit plant growth and/or to conferprotection of the plant against a plant pest. The additionalagricultural agent may be a microbial agent, a biological agent, or achemical agent.

In some embodiments of compositions and methods, the composition can beformulated for compatibility with a liquid fertilizer.

The formulation compatible with a liquid fertilizer can include ahydrated aluminum-magnesium silicate and at least one dispersant. Theterm “in a formulation compatible with a liquid fertilizer” as usedthroughout the specification and claims is intended to mean that theformulation is capable of dissolution or dispersion or emulsion in anaqueous solution to allow for mixing with a fertilizer for delivery toplants in a liquid formulation.

In notable embodiments, the formulation compatible with a liquidfertilizer can include bifenthrin, such as a composition comprisingbifenthrin; a hydrated aluminum-magnesium silicate; and at least onedispersant selected from a sucrose ester, a lignosulfonate, analkylpolyglycoside, a naphthalenesulfonic acid formaldehyde condensateand a phosphate ester. The bifenthrin can be preferably present in aconcentration of from 1.0% by weight to 35% by weight, moreparticularly, from 15% by weight to 25% by weight based upon the totalweight of all components in the composition. The bifenthrin insecticidecomposition can be present in the liquid formulation at a concentrationranging from 0.1 g/ml to 0.2 g/ml. The bifenthrin insecticide may bepresent in the liquid formulation at a concentration of 0.17 g/ml. Thedispersant or dispersants can preferably be present in a totalconcentration of from about 0.02% by weight to about 20% by weight basedupon the total weight of all components in the composition. In someembodiments, the hydrated aluminum-magnesium silicate may be selectedfrom the group consisting of montmorillonite and attapulgite. In someembodiments, the phosphate ester may be selected from a nonyl phenolphosphate ester and a tridecyl alcohol ethoxylated phosphate potassiumsalt.

The dispersant or dispersants can preferably be present in a totalconcentration of from about 0.02% by weight to about 20% by weight basedupon the total weight of all components in the composition.

In some embodiments, the hydrated aluminum-magnesium silicate can beselected from the group consisting of montmorillonite and attapulgite.

In some embodiments, the phosphate ester can be selected from a nonylphenol phosphate ester and a tridecyl alcohol ethoxylated phosphatepotassium salt.

Other embodiments can further include at least one of an anti-freezeagent, an anti-foam agent and a biocide.

In another aspect, the compositions may be prepared by a processfollowing the steps of combining the biological active ingredients ineffective amounts with carriers and adjuvants as described herein. Theformulated compositions can be prepared e.g. by mixing the biologicalactive agents with the formulation components in order to obtaincompositions in the form of finely divided solids, granules ordispersions. The active ingredients can also be formulated with othercomponents, such as finely divided solids, mineral oils, oils ofvegetable or animal origin, modified oils of vegetable or animal origin,organic solvents, water, surface-active substances or combinationsthereof.

In some embodiments, the components of the formulation can be dry mixed,or solid and liquid components may be blended together in a homogenizeror other suitable mixing vessel. Simple mixing of the ingredients byhomogenization may be preferable to any form of grinding. In otherembodiments, the mixture may further undergo a milling process, such asdry milling or wet milling, until suitable particle sizes ranging fromabout 1 to about 250 microns are obtained. The composition may haveparticle sizes of less than 250, less than 100 or preferably less than50 microns. In a preferred embodiment, the mixture is homogenized ormilled until 90% of the particle size (D90) is less than about 50microns.

One embodiment is directed to a composition comprising: i) the Bacillusthuringiensis RTI545 deposited as ATCC No. PTA-122161, or a mutantthereof having all the identifying characteristics thereof; and ii) atleast one formulation component selected from the group consisting ofadjuvants for an SC formulation; adjuvants for a WP formulation; andadjuvants for a WG formulation.

In another embodiment, the composition is in the form of an SC, such asone comprising water and at least one surfactant, and one or moreadditional adjuvants selected from thickeners, solvents, preservatives,antifreeze agents, pH-modifiers, and antifoam agents.

In an embodiment, the SC comprises from 1 to 20 weight % of Bacillusthuringiensis RTI545 deposited as ATCC No. PTA-122161, or a mutantthereof having all the identifying characteristics thereof; 1 to 5weight % of one or more surfactants; and optionally at least onethickener, solvent, preservative, antifreeze agent, or antifoam agent;and water. The optional thickener, solvent, preservative, antifreezeagent, or antifoam agent may each independently comprise up to about 1weight % of the SC formulation. The SC comprises water in acomplementary amount to all the other components to bring the totalcomposition to 100 weight % (qs).

The composition may be in solid form, for example a dust, powder,granule, WP or WG formulation. These formulations comprise at least onesolid carrier as described above. In embodiments, WP or WG formulationsmay comprise from about 1 to about 50 weight %, such as from 1 to 10, 5to 10, or 5 to 50, or 7 to 50, or 10 to 50 weight %, of Bacillusthuringiensis RTI545 deposited as ATCC No. PTA-122161, or a mutantthereof having all the identifying characteristics thereof; and at leastone solid carrier selected from the group consisting of maltodextrin,calcium carbonate and silica. Wettable granule formulations are similarto wettable powder formulations, except that the powder is formed intolarger granules, for example by diluting the powder in water optionallywith additional dispersant, and forming granules by agglomeration, spraydrying or extrusion.

In an embodiment, the composition may comprise from 2 to 20 weight % ofBacillus thuringiensis RTI545 deposited as ATCC No. PTA-122161, or amutant thereof having all the identifying characteristics thereof; fromabout 80 to about 90 weight % of maltodextrin, and about 0.5 to about 2weight % of silica.

In another embodiment, the composition may comprise from 5 to 60 (suchas 40%) Bacillus thuringiensis RTI545 deposited as ATCC No. PTA-122161,or a mutant thereof having all the identifying characteristics thereof;from about 30 to about 50 (such as 40%) weight % of maltodextrin, about10 to 20 (such as 16%) weight % of calcium carbonate and about 0.5 toabout 5 (such as 4%) weight % of silica.

In another embodiment, the composition comprises a wettable powder orwettable granule formulation comprising by weight %:

5-50% (such as 40%) of Bacillus thuringiensis RTI545 deposited as ATCCNo. PTA-122161, or a mutant thereof having all the identifyingcharacteristics thereof;

5-15% (such as 10%) maltodextrin;

35-45% (such as 40%) calcium carbonate; and

5-15% (such as 10%) silica.

The composition may be useful in either plant seed treatment orin-furrow applications. For seed treatment, a solution, slurry, paste,gel or moistened solid of the composition can be applied to seed usingstandard seed treatment procedures. The composition may be applied tountreated seeds or seeds that have been treated with at least oneadditional crop protection agent as described herein. Alternatively, thecomposition may also be mixed with an additional crop protection agentfor seed treatment or in-furrow applications.

In furrow applications can include treating the soil in the furrow,preferentially in proximity to the crop seeds at the time of planting,and incorporating the formulation into the soil. In furrow applicationscan include liquid or solid formulations. In some embodiments, thein-furrow applications comprise applying the composition in a foam.

In embodiments, the formulated compositions can be in the form ofconcentrates that are diluted prior to use, although ready-to-useformulations can also be made. Whereas commercial products willpreferably be formulated as concentrates, the end user will normallyemploy dilute formulations for application to the soil or plant. Thedilutions can be made, for example, with water, liquid fertilizers,micronutrients, biological organisms, oil or solvents.

In embodiments, the formulated compositions may additionally include anadditive comprising an oil of vegetable or animal origin, a mineral oil,alkyl esters of such oils or mixtures of such oils and oil derivatives.The amount of oil additive in the composition according to the inventionis generally from 0.01 to 10%, based on the spray mixture. For example,the oil additive can be added to the spray tank in the desiredconcentration after the spray mixture has been prepared. In embodiments,oil additives may comprise mineral oils or an oil of vegetable origin,for example soybean oil, rapeseed oil, olive oil or sunflower oil,emulsified vegetable oil, alkyl esters of oils of vegetable origin, forexample the methyl derivatives, or an oil of animal origin, such as fishoil or beef tallow.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentinvention and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter.

Example 1 Identification of Bacterial Isolate Bacillus thuringiensisRTI545 Through Sequence Analysis

A plant associated bacterial strain, designated herein as RTI545, wasisolated from the rhizosphere soil surrounding tall fescue grass inNorth Carolina. The genome of the strain RTI545 was sequenced, and thesequences of the 16S rRNA (SEQ ID NO.: 1) and rpoB (SEQ ID NO.: 2) genesof the RTI545 strain were compared to those of other known bacterialstrains in the NCBI and RDP databases using BLAST; this placed strainRTI545 within the Bacillus cereus/thuringiensis/anthracis clade. Furtherphylogenetic analysis of the RTI545 strain and relevant Bacillus specieswas performed using Bootstrap consensus trees (1000 replicates) on therpoB gene. The consensus tree for the rpoB gene is shown in FIG. 2. Ascan be seen in FIG. 2, the RTI545 strain forms a separate branch in theBacillus cereus/thuringiensis/anthracis clade. The differences insequence for the rpoB gene at the DNA level indicate that RTI545 is anew strain falling within the Bacillus cereus/thuringiensis/anthracisclade. Additional sequence analysis revealed that the RTI545 strainlacks the genes for crystal proteins (cry genes) often found in B.thuringiensis strains.

In addition, whole genome sequence analysis was performed to compare theRTI545 strain with closely related strains of the Bacillus species usingboth MUMmer- and BLASTn-based Average Nucleotide Identity (ANI)calculations (Richter M, & Rosselló-Móra R (2009) Shifting the genomicgold standard for the prokaryotic species definition. Proc Natl Acad SciUSA 106(45):19126-31) and UNIPEPT analysis (Mesuere, B., Debyser, G.,Aerts, M., Devreese, B., Vandamme, P. and Dawyndt, P. (2015), TheUnipept metaproteomics analysis pipeline. Proteomics, 15: 1437-1442.doi:10.1002/pmic.201400361) to confirm its phylogenetic classification.The results of the MUMmer and BLASTn based ANI calculations are shown inTable I below. Both the ANI and UNIPEPT (data not shown) analysesrevealed a significant degree of sequence similarity between RTI545 andpublished sequences of strains indicated as both B. thuringiensis and B.cereus. The highest sequence similarity to a recognized type strain wasto the recognized type strain B. thuringiensis Berliner ATCC10792.Again, the differences in whole genome sequence from those previouslypublished indicate that RTI545 is a new Bacillus thuringiensis strainfalling within the Bacillus cereus/thuringiensis/anthracis clade.

TABLE I Sequence analysis (both MUMmer and BLASTn based ANIcalculations) comparing strain RTI545 with relevant Bacillus speciesstrains. B. thuringiensis RTI545 ANI ANI Strain (BLAST) (MUMmer) B.cereus UW85 98.48 98.91 B. cereus CMCC P0011 97.59 98.11 B. cereus CMCCP0021 97.59 98.11 B. thuringiensis Berliner ATCC 10792* 97.51 98.17 B.thuringiensis Bt407 97.32 98.26 B. thuringiensis subsp. chinensis CT-4397.21 98.17 B. thuringiensis YBT-1518 97.18 98.11 B. cereus ATCC 14579*95.92 96.78 B. cereus B4264 95.84 96.70 B. thuringiensis subsp.tolworthi: Pasteur 95.99 96.66 B. bombysepticus Wang 95.86 96.52 B.thuringiensis subsp. kurstaki HD 1 95.57 96.37 B. cereus ATCC 4342 91.0192.02 B. cereus 03BB87 91.01 92.04 B. anthracis Ames A0462 90.67 91.71B. anthracis Sterne 90.65 91.71 B. weihenstephanensis DSM11821 88.6490.04 B. mycoides Rock 1-4 81.51 86.11 B. cytotoxicus NVH 391-98 80.0185.31 B. subtilis BAB-1 66.74 83.22 B. subtilis 168 66.70 85.58 B.amyloliquefaciens (B. velezensis) FZB42 66.22 85.34 B. velezensis YAUB9601-Y2 66.21 85.15 Enterobacter 638 63.92 82.06 P. furiosus DSM363862.16  0.00 Note: *indicates recognized type strains for both Bacillusthuringiensis and Bacillus cereus.

The strain of RTI545 was deposited on 12 May 2015 under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure at the American TypeCulture Collection (ATCC) in Manassas, Va., USA and bears the PatentAccession No. PTA-122161.

RTI545 genomic 165 rDNA 1 (SEQ ID NO: 1)AGAAAGGAGGTGATCCAGCCGCACCTTCCGATACGGCTACCTTGTTACGACTTCACCCCAATCATCTGTCCCACCTTAGGCGGCTGGCTCCAAAAAGGTTACCCCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACGGTTTTATGAGATTAGCTCCACCTCGCGGTCTTGCAGCTCTTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTTAATGATGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCTCCCGAAGGAGAAGCCCTATCTCTAGGGTTTTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAACTTCAGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGTGTCAGTTACAGACCAGAAAGTCGCCTTCGCCACTGGTGTTCCTCCATATCTCTACGCATTTCACCGCTACACATGGAATTCCACTTTCCTCTTCTGCACTCAAGTCTCCCAGTTTCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAGAAACCACCTGCGCGCGCTTTACGCCCAATAATTCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCAGCTTATTCAACTAGCACTTGTTCTTCCCTAACAACAGAGTTTTACGACCCGAAAGCCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGACGCGGGTCCATCCATAAGTGACAGCCGAAGCCGCCTTTCAATTTCGAACCATGCAGTTCAAAATGTTATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTATGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACTTCTTGAGAGCAAGCTCTCAATCCATTCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCATCCTGAGCCAGGATCAAAC

RTI545 rpoB gene (SEQ ID NO: 2)TTGACAGGTCAACTAGTTCAATACGGACGCCACCGCCAACGAAGAAGTTATGCCCGTATTAGTGAAGTATTAGAGTTACCAAATCTTATCGAAATTCAAACCTCTTCTTATCAGTGGTTTCTTGATGAGGGTTTGCGAGAAATGTTCCAAGACATTTCTCCGATTGAAGACTTTACGGGAAATCTATCGCTTGAATTTATCGACTACAGCTTAGGTGAACCTAAATACTCTGTAGACGAATGCAAAGAGCGTGATGTGACGTATGCAGCACCACTTCGTGTAAAAGTGCGTCTAATCAACAAGGAAACTGGTGAAGTAAAAGAACAAGATGTGTTCATGGGAGATTTCCCACTCATGACAGAGACTGGAACATTCGTAATTAACGGTGCAGAACGTGTTATCGTTTCCCAGTTAGTTCGCTCTCCAAGCGTATACTATAGTGGCAAAGTGGATAAAAACGGAAAACGTGGTTTTACTGCTACTGTAATTCCAAACCGCGGAGCTTGGTTAGAGTATGAGACAGATGCTAAGGATGTTGTATATGTGCGTATTGACCGTACGCGTAAACTTCCTGTAACTGTTTTGTTACGCGCATTAGGGTTTGGCTCTGATCAAGAAATCACCGAGCTTTTAGGTGATAACGAATACTTAAGCAACACATTAGAAAAAGACAACACAGATAGTACAGAAAAAGCATTGCTTGAAATTTATGAGCGTCTACGTCCTGGTGAACCACCAACAGTAGAAAATGCTAAGAGCTTACTTGTGTCTCGTTTCTTCGATCCAAAGCGCTACGATTTAGCAAATGTAGGTCGCTATAAGATCAACAAGAAGTTACACATTAAAAACAGATTGTTTAATCAACGTTTAGCTGAAACATTAGTGGATCCAGAAACTGGTGAAATTTTAGCGGCAGAAGGAACAATCTTAGATCGTCGTACACTTGATCGCATTTTACCTTACTTAGAGAAAAACATTGGATTCAAAACAGCGAAACCAATGGGTGGAGTGGTAGAAGGCGATGTTGAGCTGCAATCTATTAAGATTTATGCTCCTGAGTCGGAAGGCGAACGTGTAATTAATGTAATTGGTAATGCAAATATTACTCGTGATGTGAAACACATCACACCAGGTGATATCCTTGCTTCTATCAGTTACTTCTTCAACCTACTATACAAAGTAGGGGATACAGATGATATTGACCATTTAGGAAACCGTCGTCTGCGTTCTGTTGGAGAACTATTACAAAATCAATTCCGTATCGGTCTTTCTCGTATGGAACGTGTTGTTCGTGAGAGAATGTCGATCCAAGATACAAATGCAATTACACCACAGGCGCTAATTAATATTCGTCCTGTTATTGCATCTATTAAAGAGTTCTTCGGAAGTTCTCAGTTATCTCAGTTCATGGACCAAACAAATCCATTAGCAGAGTTAACTCACAAACGAAGACTATCTGCATTAGGACCTGGTGGTTTAACGCGTGAGCGCGCAGGCTTTGAAGTACGTGACGTTCATTACTCCCACTACGGTCGTATGTGTCCGATTGAAACACCAGAGGGACCAAACATCGGTTTGATTAACTCATTATCTTCGTTCGCGAAAGTAAATGAGTTTGGTTTCATTGAAACACCATATCGTCGTGTTGACCCAGAAACTGGTCTTGTAACAGGGCATGTTGATTATTTAACAGCAGATGAAGAAGATAACTATGTTGTAGCCCAAGCGAATATGAAATTATCTGATGAAGGTGAATTCCTAAGTGAAGATATCGTAGCTCGTTTCCGTGGTGAAAACATTGTCACAAATAGAGAACGCATCGACTACATGGATGTATCTCCAAAACAAGTAGTGTCGGCAGCGACAGCTTGTATTCCGTTCTTAGAAAACGATGACTCTAACCGCGCACTTATGGGAGCGAACATGCAACGTCAGGCGGTTCCGTTAATGAATCCGGAATCTCCGATTGTAGGTACAGGTATGGAGTACGTATCAGCAAAAGACTCAGGTGCTGCAGTAATCTGTAAACATCCTGGTGTTGTTGAGCGCGTAGAAGCACGTGAAGTTTGGGTACGTCGCTATGTAGAAGTTGACGGTCAAACAGTAAAAGGCGACTTAGATCGCTACAAAATGCAAAAATTCATTCGTTCTAACCAAGGAACTTGTTACAACCAACGTCCAATCGTAAGTGTTGGAAATGAAGTTGTAAAAGGTGAAATCCTTGCGGATGGTCCTTCTATGGAATTAGGTGAACTAGCACTTGGACGTAACGTGCTTGTTGGCTTCATGACTTGGGACGGTTATAACTACGAGGATGCGATCATCATGAGTGAGCGCCTTGTAAAAGATGATGTGTACACTTCTATTCATATTGAAGAATATGAATCAGAAGCTCGTGATACGAAGCTTGGACCAGAAGAAATTACACGTGACATTCCAAATGTTGGGGAAGACGCATTACGTAACCTTGACGAGCGCGGTATCATTCGCGTTGGTGCTGAAGTAAAAGATGGAGATTTACTTGTTGGTAAAGTAACACCTAAAGGTGTAACAGAATTAACAGCTGAAGAACGTCTATTACATGCTATCTTTGGAGAAAAAGCGCGTGAAGTACGTGATACATCACTACGTGTACCACACGGTGGTGGCGGTATTATCTTAGACGTAAAAGTATTCAACCGTGAAGATGGCGATGAATTGCCACCAGGCGTGAATCAACTTGTACGTGCATATATCGTTCAAAAACGTAAAATTTCTGAAGGTGACAAGATGGCCGGACGTCACGGTAACAAAGGTGTTATTTCTCGTATTTTACCAGAAGAAGATATGCCTTACTTACCAGACGGTACGCCAATCGATATCATGTTAAACCCATTAGGGGTACCATCTCGTATGAATATCGGTCAGGTATTAGAGCTTCATCTTGGTATGGCAGCAAGATACCTGGGCATTCACATTGCAACACCAGTATTCGATGGTGCTCGTGAGGAAGATGTTTGGGGCACAATTGAAGAAGCTGGTATGGCAAATGACGCGAAAACAATCCTGTATGACGGACGTACTGGTGAACCATTCGATAACCGCGTATCTGTTGGTGTCATGTATATGATCAAACTTGCGCACATGGTTGACGATAAACTTCATGCTCGTTCTACTGGACCATACTCACTTGTAACGCAGCAACCTCTTGGAGGTAAAGCTCAGTTCGGTGGACAGCGTTTCGGTGAGATGGAGGTTTGGGCACTTGAAGCTTACGGTGCTGCTTATACTCTTCAAGAAATCTTAACAGTGAAGTCTGATGATGTTGTTGGACGTGTTAAGACTTATGAAGCAATTGTTAAAGGCGAAAATGTTCCAGAACCAGGCGTTCCTGAATCATTCAAAGTATTGATTAAAGAGCTGCAAAGTTTAGGTATGGACGTTAAAATGATGTCTAGCGACGATACAGAAATTGAAATGCGTGATACAGAAGATGACGATGATCATCAATCAGCAGATAAATTGAATGTCGAAGTTGAGACAACTAAGGAATAA

Example 2 Anti-Microbial Properties of Bacillus Thuringiensis RTI545Isolate

The antagonistic ability of strain RTI545 against major plant pathogenswas measured in plate assays. A plate assay for evaluation of antagonismagainst plant fungal pathogens was performed by growing the bacterialisolate and pathogenic fungi side by side on 869 agar plates at adistance of 3-4 cm. Plates were incubated at room temperature andchecked regularly for up to two weeks for growth behaviors such asgrowth inhibition, niche occupation, or no effect. In the case ofscreening for antagonistic properties against bacterial pathogens, thepathogen was first spread as a lawn on 869 agar plates. Subsequently, 20μl aliquots of a culture of RTI545 were spotted on the plate. Plateswere incubated at room temperature and checked regularly for up to twoweeks for an inhibition zone in the lawn around the positions wereRTI545 had been applied. A summary of the antagonism activity is shownin Table II below.

TABLE II Antagonistic properties of Bacillus thuringiensis RTI545isolate against major plant pathogens Anti-Microbial Assays RTI545Fungal Alternaria solani + Pathogens Aspergillus flavus ++ Botrytiscinerea ++ Cercospora sojina + Fusarium colmorum +− Fusarium graminearum++ Fusarium oxysporum ++ Fusarium oxysporum f. sp. Cubense + Fusariumvirguliforme ++ Glomerella cingulata +++ Magnaporthe grisea ++ Monilinafructicola +++ Phytophthora capsici −/+− Pythium sylvatium + Pythiumaphandermatum + Rhizoctonia solani ++ Sclerotinia homeocarpa ++Sclerotinia sclerotiorum +++ Stagonospora nodorum +++ Bacterial Erwiniaamylovora + Pathogens Pseudomonas syringae pv. tomato − Xanthomonaseuvesicatoria − +++ very strong activity, ++ strong activity, +activity, +− weak activity, − no activity observed

Example 3 Phenotypic Traits of Bacillus Thuringiensis RTI545 Isolate

In addition to the antagonistic properties, various phenotypic traitswere also measured for the Bacillus thuringiensis RTI545 strain and thedata are shown below in Table III. The assays were performed accordingto the procedures described in the text below Table III.

TABLE III Phenotypic Assays: phytohormone production, acetoin and indoleacetic acid (IAA), and nutrient cycling of RTI545 isolate.Characteristic Assays RTI545 Acid Production (Methyl Red) + AcetoinProduction (MR-VP) +++ Chitinase − Indole-3-Acetic Acid production −Protease activity +++ Phosphate Solubilization +− Phenotype Cream,somewhat dry with texture/wrinkles in colony, and the strain exhibitsrobust growth at 10° C. +++ very strong, ++ strong, + some, +− weak, −none observed

Acid and Acetoin Test. 20 μl of a starter culture in rich 869 media wastransferred to 1 ml Methyl Red—Voges Proskauer media (Sigma Aldrich39484). Cultures were incubated for 2 days at 30° C. 200 rpm. 0.5 mlculture was transferred and 50 μl 0.2 g/l methyl red was added. Redcolor indicated acid production. The remaining 0.5 ml culture was mixedwith 0.3 ml 5% alpha-napthol (Sigma Aldrich N1000) followed by 0.1 ml40% KOH. Samples were interpreted after 30 minutes of incubation.Development of a red color indicated acetoin production. For both acidand acetoin tests non-inoculated media was used as a negative control(Sokol et al., 1979, Journal of Clinical Microbiology. 9: 538-540).

Indole-3-Acetic Acid. 20 μl of a starter culture in rich 869 media wastransferred to 1 ml 1/10 869 Media supplemented with 0.5 g/l tryptophan(Sigma Aldrich T0254). Cultures were incubated for 4-5 days in the darkat 30° C., 200 RPM. Samples were centrifuged and 0.1 ml supernatant wasmixed with 0.2 ml Salkowski's Reagent (35% perchloric acid, 10 mMFeCl₃). After incubating for 30 minutes in the dark, samples resultingin pink color were recorded positive for IAA synthesis. Dilutions of IAA(Sigma Aldrich 15148) were used as a positive comparison; non inoculatedmedia was used as negative control (Taghavi, et al., 2009, Applied andEnvironmental Microbiology 75: 748-757).

Phosphate Solubilizing Test.

Bacteria were plated on Pikovskaya (PVK) agar medium consisting of 10 gglucose, 5 g calcium triphosphate, 0.2 g potassium chloride, 0.5 gammonium sulfate, 0.2 g sodium chloride, 0.1 g magnesium sulfateheptahydrate, 0.5 g yeast extract, 2 mg manganese sulfate, 2 mg ironsulfate and 15 g agar per liter, pH7, autoclaved. Zones of clearing wereindicative of phosphate solubilizing bacteria (Sharma et al., 2011,Journal of Microbiology and Biotechnology Research 1: 90-95).

Chitinase Activity.

10% wet weight colloidal chitin was added to modified PVK agar medium(10 g glucose, 0.2 g potassium chloride, 0.5 g ammonium sulfate, 0.2 gsodium chloride, 0.1 g magnesium sulfate heptahydrate, 0.5 g yeastextract, 2 mg manganese sulfate, 2 mg iron sulfate and 15 g agar perliter, pH7, autoclaved). Bacteria were plated on these chitin plates;zones of clearing indicated chitinase activity (N. K. S. Murthy &Bleakley., 2012. “Simplified Method of Preparing Colloidal Chitin Usedfor Screening of Chitinase Producing Microorganisms”. The InternetJournal of Microbiology. 10(2)).

Protease Activity.

Bacteria were plated on 869 agar medium supplemented with 10% milk.Clearing zones indicated the ability to break down proteins suggestingprotease activity (Sokol et al., 1979, Journal of Clinical Microbiology.9: 538-540).

Example 4 Effects of Bacillus thuringiensis Isolate RTI545 on Corn SeedGermination

The effect of vegetative cells of the bacterial isolate RTI545 on cornseed germination was determined as described below.

Assays with vegetative cells of RTI545 were performed using seeds fromcorn. RTI545 was plated onto 869 media from a frozen stock and grownovernight at 30° C. An isolated colony was taken from the plate andinoculated into a 50 mL conical tube containing 20 mL of 869 broth. Theculture was incubated overnight with shaking at 30° C. and 200 RPM. Theovernight culture was centrifuged at 10,000 RPM for 10 minutes.Supernatant was discarded and pellet was resuspended in MgSO₄ to wash.The mixture was centrifuged again for 10 minutes at 10,000 RPM. Thesupernatant was discarded and the pellet was resuspended in ModifiedHoagland's solution. The mixture was then diluted to provide an initialconcentration. From this, dilutions of the RTI545 culture were made tohave a final concentration of 2×10⁷ cfu/ml. For the seed germinationexperiments on corn, plant growth containers were labeled with RTI545 orcontrol. Ten (10) seeds were placed in a single container. Ten mL of theRTI545 suspension with a concentration of 2×10⁷ cfu/ml was added to thecontainers and the seeds were incubated at 21° C. in the dark. Controlcontainers contained seeds and Modified Hoagland's solution withoutadded bacteria. Images of the containers were taken after 10 to 12 days.FIGS. 3A-3B are images of the corn seedlings after 12 days grown in thepresence (FIG. 3A) and absence (FIG. 3B) of the RTI545 strain. As can beseen in the figures, the presence of the RTI545 strain resulted in asignificant growth advantage.

Example 5 Growth Effects of Bacillus thuringiensis RTI545 Isolate inCorn

The effect of application of the bacterial isolate on early plant growthand vigor in corn was determined. The experiment was performed byinoculating surface sterilized germinated corn seeds for 2 days in asuspension of 10⁸ CFU/ml of the bacterium at room temperature undershaking (a control was also performed without bacteria). Subsequently,the innoculated seeds were planted in 1 gallon pots filled with PROMIXBX which was limed to a pH of 6.5. For each treatment 9 pots were seededwith a single corn seed. Pots were incubated in the greenhouse at 22° C.with light and dark cycle of 14/10 hrs and watered twice a week asneeded.

Forty two days after planting, plants were harvested and their fresh anddry weight were measured and compared to data obtained fornon-inoculated control plants. The wet and dry weight of the corn shootbiomass was measured after 42 days growth. Wet weight of the corn shootbiomass was equal to 173.7 g for the plants inoculated with the Bacillusthuringiensis RTI545 strain versus a wet weight equal to 147.6 g for thenon-inoculated control which is a 17.7% increase in wet weight over thenon-inoculated control. Dry weight of the corn shoot biomass was equalto 16.0 g for the plants inoculated with the Bacillus thuringiensisRTI545 strain versus a dry weight equal to 12.4 g for the non-inoculatedcontrol which is a 29% increase in dry weight over the non-inoculatedcontrol. As can be discerned from the significant increase in both wetand dry biomass, the presence of the RTI545 strain resulted in asignificant growth advantage.

Example 6 Activity of Bacillus thuringiensis RTI545 Isolate AgainstInsects

The ability of the Bacillus thuringiensis RTI545 strain to antagonizeWestern Plant Bug (WPB), Lygus hesperus, and Southern Corn Rootworm(SCRW), Diabrotica undecimpunctata howardi, was evaluated in in vitroassays.

For the assays Bacillus thuringiensis RTI545 was grown for 7 hours in 5ml of 869 medium, at 200 rpm, and at 30° C. Subsequently, a smallportion of the pre-culture was diluted 100-fold into 869 medium andgrown for 17 hours at 150 rpm at 30° C. The entire bacterial culture wasused in all bioassays. As a biological control, Bacillus thuringiensissubsp. kurstaki HD-1 was used according to the same protocol.

For antagonism against WPB, Bacillus thuringiensis RTI545 was evaluatedin direct spray, choice feeding, and no-choice feeding assays along withthe controls: 869 medium blank, chemical control ACEPHATE 97UP (A.I.=97%O,S-Dimethyl acetylphosphoramidothioate), biological control HD-1, andan untreated control. As expected, no significant mortality (directspray and no-choice feeding assays) or repellency (choice feeding assay)was observed for the 869 medium blank or HD-1 treatments, while thechemical control killed (direct spray and no-choice feeding assays) andrepelled (choice feeding assay) the WPB. Bacillus thuringiensis RTI545provided no significant mortality to WPB when applied in both directspray and in no-choice feeding assays; however, unexpectedly, RTI545displayed a repellent behavior at 124 hours after WPB were placed intochoice assay arenas. Specifically, when the WPB were placed into acontainer containing a treated and a non-treated food source, WPB wereobserved to be feeding only on the untreated food source (data notshown).

For antagonism against SCRW larvae, Bacillus thuringiensis RTI545 cellswere evaluated in a choice feeding assay of corn seedlings and comparedto a water control. Additional treatments compared to the water controlwere i) Bacillus thuringiensis subsp. kurstaki HD-1 (HD-1), ii) chemicalcontrol CAPTURE LFR (A.I.=17.15% bifenthrin), and iii) 869 medium.Filter paper was cut in half and each section taped down within a 100 mmpetri dish, making sure that each of the two halves of filter paper didnot touch. A total volume of 0.65 ml of treatment was applied to thetreated filter paper half. Deionized water was applied to the untreatedside. For the untreated control, both halves of the filter paper weretreated with water only. One germinated corn seed was situated on eachmoist filter paper half. Ten second-instar larvae were placed at themidline between treated and untreated filter paper. There were 3replicates per treatment. Dishes were sealed with PARAFILM andmaintained in a dark environment at room temperature for 6 days prior toassessment. The location and number of dead larvae were recorded. Theproportion of larvae on each section of filter paper was square roottransformed to normalize distribution and statistically analyzed withANOVA. Utilization of post-hoc Tukey HSD test was used to determine ifdifferences between untreated and treated filter paper were significant(α=0.10).

An image of the plate assay with the RTI545 cells after 6 days is shownin FIG. 4, and the data from all of the plate assays are summarized inTable IV below. As was observed in the assay above for WPB, the RTI545unexpectedly repelled, but did not kill the SCRW larvae. As can be seenin FIG. 4 and Table IV, the RTI545 cultures were excellent at repellingthe SCRW larvae; 100% of the larvae were present on the water-treatedhalf of the filter paper and none of the larvae on the RTI545 treatedpaper. In contrast, the larvae were statistically evenly divided betweentreatment and water control for the HD-1 strain. The bifentrin chemicalcontrol resulted in about 19% of the larvae present on the CAPTURELFR-treated filter paper. The results show that the RTI545 strain wasunexpectedly superior to a chemical insecticide at repelling the insectsfrom the corn seed, but did not kill the insects.

TABLE IV Percent of living SCRW larvae located on the treatment andwater-treated halves of the filter paper after 6 days in a choicefeeding assay on corn. Percent Alive on Treatment Filter CAPTURE PaperRTI545 HD-1 LFR 896 Medium Water Treated  0.00 46.66 19.44 34.81 56.66Water 100.00  53.33 80.55 65.18 43.33 P-Value  <0.001 0.671  <0.001  0.012   0.350 Note: Statistical analyses were conducted on the squareroot transformed proportion of larvae on treated and untreated halves ofthe filter paper.

The choice feeding assay for SCRW larvae using corn seedlings wasrepeated HD-1 strain and RTI545, Unlike HD-1, RTI545 does not containgenes for production of crystal proteins (represented as “Cry”).Repellency was measured using a choice in vitro plate feeding assay ofcorn seedlings by SCRW larvae and scored after 72 hours as thepercentage of SCRW larvae on the non-treated side of the plate. In thecase of no repellency, an equal distribution of 50% on both the treatedand non-treated half of the plate would be expected, indicating 0%repellency. The results of this assay are shown in Table V below. Inthis experiment, the Bacillus thuringiensis RTI545 and Bacillusthuringiensis subsp. kurstaki HD-1 (HD-1), were evaluated in comparisonto a water control after 3 days in the assay. Again, the RTI545 cultureswere excellent at repelling the SCRW larvae; 96% of the larvae werepresent on the water-treated half of the filter paper and only 4% on thehalf containing the RTI545 cells. In contrast, the larvae were againstatistically evenly divided between treatment and water control for theHD-1 strain (53% on non-treated side of plate).

TABLE V Percent of living SCRW larvae located on the non- treated halfof filter paper after 3 days in a choice feeding assay on corn. % oflarvae on Bacterial Strain Cry non-treated side Bacillus thuringiensisRTI545 − 96 Bacillus thuringiensis subsp. kurstaki HD-1 + 53

The choice feeding assay for SCRW larvae using corn seedlings was alsorepeated to compare RTI545 to another Bacillus thuringiensis strain FD30and kanosamine. Based on genome sequence data, the putative kanosaminebiosynthesis pathway is found in RTI545 but not in FD30. Repellency wasmeasured using a choice in vitro plate feeding assay of corn seedlingsby SCRW larvae and scored after 72 hours as the percentage of SCRWlarvae on the non-treated side of the plate. In the case of norepellency, an equal distribution of 50% on both the treated andnon-treated half of the plate would be expected, indicating 0%repellency. The results of this assay are shown in Table VI below.

TABLE VI Mean percent of SCRW larvae located on the untreated andtreated portions of the filter paper at 3 days after introduction intothe experimental arena. B. t. strain Kanosamine 10 100 100 μg/ml + 869Assay Untreated RTI545 FD30 μg/ml μg/ml FD30 media Treated 46.67  16.67 40.00  16.67  16.67  6.67 30.0  Untreated 53.33  83.33  60.00  83.33 83.33  93.33  70.0  p-value  0.612 <0.001  0.072  0.003  0.003  0.002 0.01

As summarized in Table VI, RTI545 provides a repellent effect to SCRWlarvae when placed on the midline between treated and non-treated filterpapers. FD30 (Bacillus thuringiensis) does not have the same overalleffect. SCRW avoidance was observed when kanosamine was combined withthe FD30 strain. In two separate bioassays, repellent effects bykanosamine, at all dilution rates between 0.1 μg/ml and 100.0 μg/ml,were observed at 24 h. In one test, kanosamine treated filter paper at10 and 100 μg/ml provided z 80% avoidance response to SCRW. At 3 d,minimal feeding damage was observed on corn located on the kanosaminetreated side. Conversely, FD30 had a non-statistical 20% difference inthe number of larvae located on the treated and untreated filter paperat 3 d; noticeable feeding damage to corn was seen on both sides of theFD30 choice assay. In a second test, filter paper treated with 30.0ug/ml of kanosamine provided complete repellency out to 5 days (data notshown). Based on these results, the ability of RTI545 to repel insectspecies such as WPB and SCRW may be due to its production of kanosamine.

Example 7 Activity of Bacillus thuringiensis RTI545 Isolate AgainstNematodes

The results of Example 6 suggest that repellent activity of TRI545against insect species may be due to production of compounds such askanosamine. Similarly, activity of RTI545 against nematodes may also bedue to compound(s) produced by the strain. A nematode chemotaxis assayon agar plates was conducted as described below to evaluate response ofroot knot (RKN) nematode juveniles (J2) to different concentrations ofkanosamine and RTI545 supernatant in vitro.

The test arena is shown schematically in FIG. 6. Nine-cm-diameter Petridishes were filled with 15 ml 0.75% Phytagel (including 0.1%MgSO₄.7H₂O). Magnesium Sulfate Heptahydrate MgSO₄.7H₂O was used, insteadof water agar, so that nematode tracks could be observed under amicroscope. Wells of 0.5 cm diameter which can accommodate approximately50 μl of solution were made at opposite sides of the dishes at 2 cm fromthe center. The test samples were applied in the wells and left todiffuse for 1 hour with lids on the dishes, so a gradient around thewells could be established. Then, 75-100 J₂ stage root-knot nematodessuspended in 5 μl of sterile distilled water were placed by pipette inthe center 1.5 cm-diameter circle of the plate. When the surface tensionof the water suspension was lost, the dishes were covered with lids andincubated in the dark on a leveled platform at 25° C. for 1 to 4 hours.After incubation the plates were transferred to 4° C. to stop nematodemovement for scoring at 2 h, 3 h and 5 h after setup. Three replicateswere done for each test material.

For scoring, the test arena was divided into sixteen zones, designatedas 1-8 for attractive zones and a-h for repellent zones as shown in FIG.6A. Nematodes attracted to the test substance would tend to move to thenumbered (attractive) zones, resulting in clustering along the axisparallel to the orientation of the wells. Repelled nematodes would tendto move to the lettered (repellant) zones, resulting in clustering alongthe axis perpendicular to the orientation of the wells. The chemotaxisfactor (Cf) was calculated by dividing the total number of nematodes inthe attractant zones by the total number of nematodes in the repellentzones. A Cf greater than 2 meant attraction for the nematodes, whilelower than 0.5 indicated repellence, and 0.5 to 2 was consideredneutral. The results are shown in Table VII. In this chemotaxis bioassaydistilled water was found to be neutral at all evaluation time points(Cf between 0.5 and 2.0). Acetic acid at 1% showed repellency to RKN J2(Cf<0.5) at 3 h and 5 h from initiation of the test. The Cf for aceticacid at early evaluation time point of 2 h was neutral (data not shown),probably as a result of slow diffusion of the chemical into the Phytagelmedium and/or delayed nematode response to the chemical. FIG. 6B is aphotograph of an assay of kanosamine tested at 100 μg/ml, wherein dotsrepresenting nematode locations indicate neutral distribution. FIG. 6Cis a photograph of an assay of RTI545 supernatent tested at 100%strength, wherein dots representing nematode locations indicaterepellant distribution.

TABLE VII The chemotaxis of root knot nematode (Meloidogyne spp.) in thepresence of RTI545 supernatant. Cf (chemotaxis factor) Treatment at 3 hat 5 h kanosamine 1 μg/ml 1.0   1.0  kanosamine 10 μg/ml 0.6   0.7 kanosamine 100 μg/ml 0.6   0.7  Water 1.0   1.0  Acetic Acid (1%) 0.45 0.4  RTI545 supernatant 1% 0.5   0.4  RTI545 supernatant 10% 0.2   0.1 RTI545 supernatant 25% 0.1   0.1  RTI545 supernatant 50% 0.003 0.03RTI545 supernatant 100% 0.003 0.01 869 medium 10% 0.1   0.01 869 medium50% 0.1   0.06 869 medium 100% 0.03  0.02

All three tested concentrations of kanosamine (1, 10 and 100 μg/ml) didnot show repellent properties in the assay. The Cf factor wasconsistently higher than 0.5 for all tested kanosamine rates and alltested time points. All tested rates of RTI545 supernatant (1%, 10%,25%, 50% and 100%) and all rates of 869 medium (10%, 25%, 50% and 100%)act as a repellent (Cf<0.5) starting from 2 h of the initiation of theassay. Such quick response of J2 nematodes suggests that the factorresponsible for nematode behavior easily diffuses and establishes agradient. In the case of the 869 medium there was no clear dose responseto tested rates. However, in the case of RTI545 supernatant, clearresponse to tested rates was observed: higher rates of supernatantresulted with stronger repulsion of nematodes (lower Cf). However, theresults for RTI545 repellency are non-conclusive due to strong activityof 869 medium of the assay.

The absence of repellency of kanosamine to nematodes is in contrast tothat observed for insect assays. Nematode repellency observed for RTI545appears to be due to some other factor than kanosamine.

The compounds produced during overnight culture of RTI545 (Bacillusthuringiensis) strain and the pure compound kanosamine were evaluated invitro to characterize their potential effect on hatching of root knotnematode (RKN) eggs (Meloidogyne incognita/hapla).

Bacterial Supernatants:

To obtain supernatant for the assay, one loop (10 l) of the RTI545strain was grown for 16 h in 5 ml of 869 medium at 200 rpm at 30° C. Thefollowing day, the optical density (OD) of 1:100 dilutions was measuredat 600 nm to estimate the volume needed for inoculation of fermentationflasks. The bacterial culture was started in 250 ml fermentation flasksin 25 ml of 869 medium at an initial OD of 0.01. Bacteria were grownovernight (for 16 h) at 200 rpm at 30° C. Two ml of bacterial culturewas saved to measure the OD and colony forming units (CFU). Theremaining culture was centrifuged (2500 rpm, 15 min) and the supernatantwas filter-sterilized through a 0.22 μm filter. The hatching assay wasinitiated within 4 h from the supernatant collection. The supernatantwas kept at 4° C. until the initiation of the bioassay.

Kanosamine (10 mg) was dissolved in 1 ml of deionized water to obtain 10mg/ml stock solution. To obtain 200 μg/ml concentration, 20 μl ofconcentrated stock (10 mg/ml) was added to 990 ml of water. Serialdilutions were then made to create concentrations of 20 μg/ml and 2μg/ml of kanosamine respectively.

A mixed culture of root knot nematodes (Meloidogyne incognita and M.hapla) was used. Nematode eggs were extracted from tomato roots bybleaching and cleaned using an Opti-prep centrifugation step followed bytwo washes in water. Before setting up the hatching assay, thepercentage of early (eggs with a visible embryo) and late eggs (eggswith differentiated juveniles inside; J1 or J2 stages) were establishedby counting the eggs under a microscope. Only fresh eggs (collected atthe day of the initiation of the assay) were used for the bioassay.

The assay was performed in 24-well tissue culture plates. In each well75 μl of egg solution in 2% methyl cellulose (approximately 100 eggs perwell) was mixed with 75 μl of antibiotic solution (300 mg/Lstreptomycin+300 mg/L penicillin) and 150 l of each treatment.Antibiotics were suspended in sterile distilled water. The finalconcentration of antibiotics in testing plates was 75 mg/L of penicillinand 75 mg/L of streptomycin. All treatments contained 2% methylcellulose (RKN eggs were suspended in methyl cellulose prior to exposureto treatment). The addition of methyl cellulose increases the accuracyof adding the same number of inoculum to each treatment. The treatmentswere set up in 6 replications. Each plate was covered, wrapped inaluminum foil and placed in an incubator set at 25° C. The numbers ofhatched juveniles in each well were counted under a stereomicroscope at7 days and 14 days from initiation of hatching. For each time point andtreatment, the percent hatching was calculated according to the formula:

${\%{hatching}} = {\frac{{number}{of}J2{in}{well}}{\begin{matrix}{{number}{of}{eggs}{present}{in}} \\{{the}{well}{at}{the}{beginning}{of}{the}{assay}}\end{matrix}}*100\%}$

Mean percent hatching of root-knot nematode eggs after 7 and 14 days areshown in Table VIII. All treatments were supplemented with antibiotics(75 mg/L of penicillin and 75 mg/L of streptomycin) to preventcontamination. Data are means from 6 replicates± standard deviation ofthe means.

TABLE VIII Impact of treatments on nematode egg hatching % % Hatching %Hatching Treatments after 7 days after 14 days  1 RTI545 - 2.5% (v/v)supernatant  5.2 ± 1.5 9 ± 4  2 RTI545 - 12.5% (v/v) supernatant  6.9 ±3.3 9.5 ± 5.3  3 RTI545 - 25% (v/v) supernatant  5.6 ± 1.8 9.6 ± 5.1  4RTI545 - 50% (v/v) supernatant  5.2 ± 2.5 6.4 ± 2.3  5 kanosamine - 1μg/ml 35.9 ± 5.2 40.1 ± 6.8   6 kanosamine - 10 μg/ml 39.4 ± 3   42.1 ±12.1  7 kanosamine - 100 μg/ml 32.4 ± 4.8 41.6 ± 6.8   8 abamectin 0.1ppm 24.4 ± 2.9 27.8 ± 5.7   9 abamectin 1.0 ppm  4.9 ± 0.9 8.5 ± 1.6 10medium 869 22.5 ± 3.5 28.3 ± 3.7  11 water without antibiotics 22.2 ±2.6 30.3 ± 3.9  12 water 21.3 ± 1.9 30.9 ± 5.4 

The percentage of early and late eggs collected for the assay was 41%and 59%, respectively. The antibiotics, lower rate of Agrimek® (0.1 ppm)and 869 media blank did not have a significant effect on egg hatching.The chemical standard Agrimek® inhibited egg hatching in vitro. The rateof 1 ppm caused 72% egg hatching inhibition after 14 days. The hatchingin any rate RTI545 supernatant and in the high rate of Agrimek® (1 ppm)was significantly lower than in the water control. A dose response wasnot observed among the tested RTI545 rates (2.5%-50%), as well as allthese rates were comparable to the chemical standard Agrimek®. Incontrast, the hatching rate of eggs exposed to various concentrations ofkanosamine (1, 10 and 100 μg/ml) was higher than in water control.

RTI545 supernatant from overnight culture on 869 medium at all testedrates significantly inhibited egg hatching. Similar results wereobserved when RTI545 supernatant was collected from 3 days culture grownon the same medium (data not shown). The compounds responsible for egghatching inhibition are present in cultures that were growth overnightand for 3 days.

Kanosamine had a positive effect on egg hatching. The hatching rate ofeggs exposed to kanosamine was up to 80% higher than hatching rate ofeggs incubated in water control after seven days. These results are inagreement with biochemical properties of kanosamine. Kanosamine wasidentified as chitin synthesis inhibitor (Janiak and Milewski, 2001).Nematode egg shell is composed of chitin and egg hatching involveschitin degradation—the opposite process to chitin synthesis.

While not intending to be bound by any theory, the results from theassays suggest that the anti-nematode activity of RTI545 is not due tokanosamine. The different behavior of RTI545 supernatants and 869 mediaextracts in the repellency and egg hatching assays suggests that RTI545produces an as-yet unidentified compound that provides anti-nematodeperformance.

Example 8 Effects on Growth and Yield by Treating Corn and Soybean Seedwith Bacillus thuringiensis RTI545

Experiments were performed to determine the effect on growth and yieldunder insect pressure by treating corn and soybean seed with spores ofB. thuringiensis RTI545, in combination with a chemical insecticide.

The effects on one or more of growth, yield, and control of the cornpests wireworm and seed maggot were measured in field trials inWisconsin. Additional experiments were performed in the greenhouse tomeasure the effect on early plant growth in the presence of wireworm.The experiments were performed as described below.

Formulations:

A B. thuringiensis RTI545 spore concentrate (1.0×10¹⁰ cfu/ml) in waterwas applied at an amount of 1.0×10⁶ cfu/seed.

MAXIM (SYNGENTA CROP PROTECTION, INC) was applied to seed at 0.0064 mgA1/kernel (A.I.=fludioxonil).

APRON XL (SYNGENTA CROP PROTECTION, INC) was applied to seed accordingto manufacturer label (A.I.=mefenoxam).

PONCHO 250, PONCHO 500 VOTIVO, and PONCHO 1250 VOTIVO (BAYER CROPSCIENCE) were each applied to seed according to manufacturer label(PONCHO A.I.=clothianidin and VOTIVO A.I.=Bacillus firmus 1-1582).

In the first field trial experiment, corn seeds were treated withslurries containing: 1) chemical control MAXIM+APRON XL (referred to as“FC”); 2) FC+ the insecticide bifenthrin 0.125 mg/seed; 3) FC+PONCHO1250 (clothianidin 1.25 mg/seed) and VOTIVO (Bacillus firmus 1-1582); 4)FC+PONCHO 250 (clothianidin 0.25 mg/seed); 5) FC+PONCHO 500(clothianidin 0.5 mg/seed) and VOTIVO (Bacillus firmus 1-1582) and; 6)FC+bifenthrin (0.125 mg/seed)+spores of B. thuringiensis RTI545.

The treated corn seed were planted in separate field trials in Wisconsinin soil infested with the insect pests wireworm and seed maggot. In onetrial, manure was added to plots to attract egg laying by Delia spp.adults. Ratings collected and analyzed were % emergence, plant stand, %wireworm damage, % seed maggot damage, plant vigor and yield. Insectfeeding damage severity was rated by visual inspection 34 days afterplanting, and plant vigor was ranked on a scale of 1 to 5, with 1 beingvery bad and 5 representing an excellent plant vigor rating.

The results are shown below in Table IX. Inclusion of the B.thuringiensis RTI545 in combination with the insecticide bifenthrinresulted in significant improvements in percent emergence, plant stand,vigor and control of both wireworm and seed maggot over seeds treatedwith bifenthrin alone. In addition, the results of the combination of B.thuringiensis RTI545 and bifenthrin were statistically equivalent to theproduct PONCHO 1250 VOTIVO at controlling wireworm and showed animprovement over this product in controlling seed maggot. These dataindicate that corn seed treatment with a combination of B. thuringiensisRTI545 and a chemical insecticide such as bifenthrin significantlyimproves insect control over inclusion of chemical insecticide alone andis superior to commercially available products for some types of insectcontrol.

TABLE IX Control of wireworm and seed maggot in corn field trials afterseed treatment with a combination of chemical insecticide and spores ofRTI545 as compared to PONCHO VOTIVO. Emerge Stand Wireworm Seed MaggotVigor TREATMENT % % % Damage % Damage 1-5 1 FC* 70 78 26 14 3 3 3 2 FC +Bifenthrin 81 85 13 11 3.8 4 4 3 FC + Poncho 1250 Votivo 91 93 6.2 5.4 44.3 4.5 4 FC + PONCHO 250 80 89 14 11 3.8 4 4 5 FC + Poncho 500 Votivo85 89 14 7.4 4 4 4 6 FC + Bifenthrin + RTI545 92 96 5.6 3.2 4.5 4.8 5*FC is the fungicide check applied to all treatments containingfludioxonil and Mefenoxam to provide disease protection

In a second field trial corn seeds were also treated with the sameslurries as the first trial containing: 1) chemical control MAXIM+APRONXL (referred to as “FC”); 2) FC+bifenthrin; 3) FC+PONCHO 1250 VOTIVO; 4)FC+PONCHO 250; and 5) FC+bifenthrin+spores of RTI545. The treated cornseed were planted in separate field trial in Wisconsin with wirewormpresent and no manure added so seed maggot was not a problem. Damage ofcorn roots from wireworm feeding was rated 41 days after planting.

The results are shown below in Table X and show similar results to TableIX above. Specifically, inclusion of the B. thuringiensis RTI545 incombination with the insecticide bifenthrin resulted in significantimprovements in percent emergence, plant stand, vigor and control ofwireworm over seeds treated with bifenthrin alone. In addition, theresults of the combination of B. thuringiensis RTI545 and bifenthrinwere statistically equivalent or superior to the product PONCHO 1250VOTIVO at controlling wireworm.

TABLE X Control of wireworm in corn field trials after seed treatmentwith a combination of chemical insecticide and spores of RTI545 ascompared to PONCHO VOTIVO. Emergence Stand Wireworrn % Vigor TREATMENT %% Damage (Root) 1-5 1 FC* 68 76 32 3 2 FC + Bifenthgrin 79 85 21 4 3FC + Poncho 1250 89 93 11 4.3 Votivo 4 FC + PONCHO 250 79 86 17 4 5 FC +Bifenthrin + 91 95  4 4.5 RTI545 *FC is the fungicide check applied toall treatments containing fludioxonil and Mefenoxam to provide diseaseprotection

The average yield in the corn field trials after seed treatment with acombination of chemical insecticide and spores of RTI545 as compared toPONCHO VOTIVO was also determined. The results are shown below in TableXI. Inclusion of the B. thuringiensis RTI545 in combination with theinsecticide bifenthrin resulted in significant improvements in yield ascompared to seeds treated with bifenthrin alone. In addition, thecombination of RTI545 and bifenthrin outperformed both PONCHO 500 VOTIVOand PONCHO 1250 VOTIVO by increasing yield 13 bushels/acre (from 180.5to 193.7 and from 185.5 to 193.7 bushels/acre, respectively)representing a 6.8% and 4.2% increase in grain yield, respectively.These data indicate that corn seed treatment with a combination ofRTI545 and a chemical insecticide such as bifenthrin significantlyimproves yield over inclusion of chemical insecticide alone, and reducesthe need for in-furrow application of larger quantities of chemicalinsecticides to control damage by insects.

TABLE XI Average yield in corn field trials after seed treatment with acombination of chemical insecticide and spores of B. thuringiensisRTI545 as compared to PONCHO VOTIVO Bushels/acre Kg/hectare AverageAverage TREATMENT LB02 LB01 N = 2 LB02 LB01 N = 2 1 FC 130.2 149.1 139.7 8,173 9359  8,767 2 FC + Bifenthrin 159.8 173.9 166.9 10,031 10,91610,476 3 FC + PONCHO 250 171.4 174.2 172.8 10,759 10,935 10,847 4 FC +PONCHO 500 VOTIVO 177 184 180.5 11,111 11,550 11,330 5 FC + PONCHO 1250VOTIVO 183.4 187.5 185.5 11,512 11,769 11,644 6 FC + Bifenthrin + RTI545191.9 195.4 193.7 12,046 12,265 12,159

The effect on growth under insect pressure by treating corn seed withspores of RTI545 was further evaluated. In a set of greenhouse studies,corn seeds were first treated with the seed treatment slurries asdescribed as follows and then planted in soil infested with the pestwireworm (10 wireworms per pot with one seed), along with a control setwhere the soil did not contain wireworm. The seed treatment slurrieswere as follows: 1) chemical control MAXIM+APRON XL (referred to as“FC”); 2) FC; 3) FC+bifenthrin (0125 mg/seed for all treatmentstreated); 4) FC+bifenthrin+RTI545 5.0×10⁶; 5) FC+bifenthrin+RTI5455.0×10⁶ and heat-treated; 6) FC+bifenthrin+RTI545 1.0×10⁶; 7) FC+RTI5455.0×10⁶; and 8) FC+PONCHO 1250. The treated seeds were evaluated forpercent emergence.

The results are shown below in Table XII. Inclusion of the B.thuringiensis RTI545 in combination with the insecticide bifenthrinresulted in 100% emergence, which was an improvement over inclusion ofbifenthrin alone and provided results equivalent to the control withoutwireworm and the FC+PONCHO 1250 chemical treatment. Wireworm feedingprunes roots causing corn plant stunting and RTI545 alone or bifenthrinwith RTI545 reduced plant stunting in surviving plants. RTI545 aloneexhibited activity on preventing plant loss but was inferior toinsecticide bifenthrin in providing early protection against stunting.However, RTI545 was more effective in preventing plant stunting as theplants grew (data not shown). These data indicate that inclusion ofspores of RTI545 in corn seed treatment, alone or in combination with achemical insecticide such as bifenthrin, significantly improves planthealth in the presence of the insect pest wireworm.

TABLE XII Emergence and growth in corn greenhouse studies in thepresense of wireworm after seed treatment with chemical insecticide andRTI545. % Plants Surviving with no stunting Emergence (based onTreatment 1 = 100% plants seeded) 1 FC no wireworm 1.00       100 2 FC0.17         0 3 FC + Bifenthrin 0.75        58 4 FC + Bifenthrin +RTI545 0.83        75 5 × 10⁶ 5 FC + Bifenthrin + RTI545 0.83        835 × 10⁶ heat 6 FC + Bifenthrin + RTI545 1.00        92 1 × 10⁶ 7 FC +RTI545 5 × 10⁶ 0.50        50 8 FC + PONCHO 1250 1.00       100 StandardDeviation  0.175389044 CV 23.13       

Experiments were performed to determine the effect on yield by treatingsoybean seed with a standard fungicidal combination of chemical activeingredients in addition to spores of B. thuringiensis RTI545, incombination with a chemical insecticide. The experiments were performedas described below.

Formulations:

A RTI545 spore concentrate (1.0×10¹⁰ cfu/ml) in water was applied at anamount of 1.0×10⁶ cfu/seed.

FC is a formulation containing fludioxonil, TPM, and mefenoxam which wasapplied to seed at 2.5 g/100 g seed (fludioxonil), 10 g/100 g (TPM), and7.5 g/100 g seed (mefenoxam).

Thiamethoxam was applied to seed at 50 g/100 g seed.

In the experiment, soybean seeds were mixed with a solutioncontaining: 1) chemical control fludioxonil/TPM/mefenoxam (“FC”); 2)FC+insecticide thiamethoxam; and 3) FC+thiamethoxam+spores of B.thuringiensis RTI545. The treated soybean seeds were planted at threesites (N=3) that had wireworm infestation, and the yield was analyzed.

The results are shown below in Table XIII. Inclusion of the RTI545spores in combination with the thiamethoxam resulted in significantimprovements in yield as compared to seeds treated with thiamethoxamalone increasing yield by 1.6 bushels/acre (from 68.2 to 69.8),representing a 2.3% increase in yield. These data indicate that soybeanseed treatment with a combination of RTI545 and a chemical insecticidesignificantly improves yield over inclusion of insecticide alone, andreduces the need for in-furrow application of larger quantities ofchemical insecticides to control damage by insects.

TABLE XIII Average yield in soybean after seed treatment with chemicalinsecticide and spores of RTI545 in addition to a standard fungicidalseed treatment (FC). Treatment Bu/A (N = 3) Kg/hectare FC 60.8 4089 FC +Thiamethoxam 68.2 4586 FC + Thiamethoxam + RTI545 69.8 4694

One possible explanation for the improved insect control though seed- orin furrow-treatment with spores of RTI545 in combination with a chemicalinsecticide is illustrated in FIG. 1. Specifically, FIG. 1A is aschematic diagram that shows insect control with a chemical insecticidealone, (i.e. without RTI545). FIG. 1A shows on the far left a plant seed(inner circle) coated with a chemical insecticide (dark band surroundinginner circle), which is surrounded by plant insect pests in the plantrhizosphere represented by horizontal marks. The middle portion of thediagram shows the sprouted plant seed with diffused insecticideprotecting the roots of the plant seed from the insect pests (protectionrepresented by the “X” marks). The far right of the diagram showsdiminished protection of the roots of the plant seed from the insectpests as the roots grow beyond the diffusion zone of the chemicalinsecticide. FIG. 1B shows how addition of Bacillus thuringiensis RTI545spores to the coating on the plant seed (or in furrow application ofRTI545 spores around the time of planting) improves insect control overuse of the insecticide coating alone. Specifically, the far right sideof the FIG. 1B diagram shows continued protection of the roots of theplant seed from the insect pests even as the roots grow beyond thediffusion zone of the chemical insecticide as a result of theestablishment of Bacillus thuringiensis RTI545 in the plant rhizosphere.

Example 9 Nematode Control by Bacillus thuringiensis RTI545 Isolate inInfested Soil

The ability of the Bacillus thuringiensis RTI545 strain to reduce thenematode infestation in soybean and potato was determined as describedbelow.

A greenhouse study was performed with soybean plants potted in soilinfected with Southern root-knot nematodes to determine the effect ofseed treatment with RTI545 spores applied at an amount of 1.0×10⁶cfu/seed. Soybean plants were potted in soil infected with live eggs ofSouthern Root-Knot nematodes (Meloidogyne incognita). Seeds treated witheach of the products PONCHO VOTIVO (BAYER CROPSCIENCE LP; A.I.=40.3%clothianidin, 8.1% Bacillus firmus 1-1582) and AVICTA COMPLETE(SYNGENTA; A.I.=11.7% thiamethoxam, 10.3% abamectin, 2.34%thiabendazole, 0.3% fludioxonil, 0.23% mefenoxam, 0.12% azoxystrobin),separately, containing chemical active ingredients at the ratesdesignated on the products labels. The data are shown in Table XIVbelow. At 63 days after initiation, there was no statistical differencein the number of nematode eggs/pot for the seed treatment with RTI545cells and the chemical combination AVICTA COMPLETE, however, the numberof nematode eggs/pot for the seed treated with RTI545 was less than thatfor the seed treated with PONCHO VOTIVO. There was no statisticaldifference in the number of juveniles/pot for any of the treatmentsafter 63 days. These data demonstrate the positive effect on nematodecontrol on soybean provided by Bacillus thuringiensis RTI545.

TABLE XIV Nematode infestation over time in soybeans after seedtreatment with RTI545 spores. Southern Root-Knot Nematode 35 DP-1 63DP-1 Egg Mass/ Eggs/ Juveniles/ Eggs/ Juveniles/ Pot Pot Pot Pot Pot No0 0 0 0 0 Nematodes Control 57 6434 2 7880 463.2 RT1545 41 6340 0 2795268.8 PONCHO 43 5540 0 6720 427.2 VOTIVO AVICTA 7 1377 0 2455.1 79.2COMPLETE CV 29.83 35.75 349.6 29.41 130.09

A greenhouse study was performed with potato plants potted in soilnaturally infected with Globodera sp. nematodes to determine the effectof treatment of the soil with Bacillus thuringiensis RTI545 cells. Inthis experiment, the effect of soil enhancement with RTI545 cells on thenumber of cysts per gram of root biomass was determined at 60 days afterinitiation of the study. Potatoes (nematode sensitive variety “Bintje”)were planted in soil infected with Globodera sp. (Control) and enhancedwith 10E⁹ cfu spores per liter soil of Bacillus thuringiensis RTI545(RTI545). The results are shown in the graph in FIG. 5. Additional soiltreatments were included in the study: VYDATE product (Vydate; DUPONT;A.I.=Oxamyl [Methoyl N′N′-dimethyl-N-[(methylcarbamoyl)oxyl)oxy]-1-thiooxamimidate), BIOACT product (BAYERCROPSCIENCES LP; A.I.=of Paecilomyces lilacinus strain 251), CAREXproduct (CAREX, NUFARM, pyridaben), and Bacillus thuringiensis spp.kurstaki HD-1). The products were applied at the rates designated on theproduct labels. As can be seen in FIG. 5, the number of cysts per gramof root biomass for the soil treated with RTI545 cells was significantlyreduced compared to all of the treatments including the productscontaining chemical active ingredients.

Example 10 Cotton Seed Treatment with Bacillus thuringiensis RTI545Inoculated with Rhizoctonia

Experiments were performed to investigate the effect on emergence, rootdisease, and yield in cotton in the presence of Rhizoctonia diseasepressure when seeds were treated with a the RTI545 strain in addition tochemical active agents for pathogen control.

Specifically, an experiment in cotton was set up as follows: 1) seed wasuntreated (UTC); 2) seed was treated with a base combination ofFludioxonil+Mefenoxam+Imidacloprid according to manufacturer label(referred to as “B”); 3) seed was treated with base plus 5×10⁺⁵ cfu/seedof RTI545 (B+RTI545); and 4) seed was treated with base plus VIBRANCE(active ingredient sedaxane; SYNGENTA CROP PROTECTION, INC) according tolabel instructions (B+VIBRANCE). Field trials were performed in Georgia.The trials were inoculated with Rhizoctonia by mixing the dried inoculumwith the seed at the time of planting to a prescribed rate to provideinfection when the seed commenced to grow. The average percent cottonemergence is presented in Table XV below.

The results in Table XV show that treating with RTI545 spores inaddition to the base resulted in significant improvement in percentemergence over that of the chemical base alone (67% versus 45% for basealone). In addition, treatment with RTI545 performed as well as the baseplus commercial product VIBRANCE with chemical active. Thus, seedtreatment with RTI545 can provide significant improvement in emergenceeven under conditions of severe pathogen pressure.

TABLE XV Cotton emergence in Rhizoctonia-inoculated soil from seedstreated with RTI545. Treatment % Emergence 1 Untreated control 23 2 B 453 B + RTI545 (5 × 10⁵ CFU/seed) 67 4 B + VIBRANCE 63

Example 11 Effects on Growth and Yield by Treating Wheat Seed withBacillus thuringiensis RTI545

Experiments were performed to determine the effect on growth and yieldunder insect pressure by wheat seed treated with spores of B.thuringiensis RTI545 alone, or in combination with one or both ofchemical fungicides and a chemical insecticide. More specifically, theeffects on growth, yield, and control of wheat pests wireworm and whitegrub were measured in field trials in Wisconsin. The experiments wereperformed as described below comparing addition of B. thuringiensisRTI545 spores to a seed and to a seed plus fungicide base, and additionof RTI545 spores in combination with 2 concentrations of theinsecticide, bifenthrin, in addition to the fungicide base.

In the field trial experiment, wheat seeds were treated with slurriescontaining: 1) chemical fungicide basedifenoconazole/tebuconazole/TPM/mefenoxan (referred to as “FC”); 2)FC+spores of B. thuringiensis RTI545 (RTI545 10⁶ cfu/g seed); 3)FC+Bifenthrin (20 g/seed); 4) FC+Bifenthrin (20 g/seed)+RTI545 10⁶ cfu/gseed; 5) FC+bifenthrin (50 g/seed); and 6) FC+bifenthrin (50g/seed)+RTI545 10⁶ cfu/g seed.

The treated wheat seed were planted in field trials in Wisconsin in soilinfested with the insect pests wireworm and white grub. Ratingscollected and analyzed were % emergence, % wireworm damage, % grubdamage, plant vigor and yield. Insect feeding damage severity was ratedby visual inspection 35 days after planting, and plant vigor was rankedon a scale of 1 to 5, with 1 being low and 5 representing highlyvigorous.

The results are shown below in Table XVI. Seed treatment with each ofthe B. thuringiensis RTI545 spores, and treatment with the RTI545 sporeswith either the fungicide base alone or in combination with theinsecticide, bifenthrin, resulted in significant improvements in percentemergence, vigor, control of wireworm and grub, and yield. In every casetested, inclusion of the RTI545 spores in the wheat seed treatmentresulted in significant improvements in growth, vigor, pest control, andyield.

TABLE XVI Control of wireworm and grub in wheat field trials after seedtreatment with a combination of chemical insecticide and spores ofRTI545. Vigor Emerge (35 DAP) % Damage Yield TREATMENT (21 DAP) % 1-5Wireworm Grub Bu/Acre Kg/Ha 1 FC* 59 3.5 26 7.5 70 4708 2 FC* + RT154572 5 10 4 82 5514 3 FC + Bifenthrin (20 g) 64 4 19 6.5 77 5178 4 FC +Bifenthrin (20 g) + 74 5 8 3 81 5472 RTI545 5 FC + Bifenthrin (50 g) 704.8 14 3.3 82 5514 6 FC + Bifenthrin (50 g) + 76 5 6 1.8 86 5784 RTI545*FC is the fungicide check applied to all treatments containingDifenoconazole + TPM + Tebuconazole + Mefenoxam to provide diseaseprotection

Example 12 Impact of Bacillus thuringiensis RTI545 Seed Treatment onGrowth and Yield of Soybeans Inoculated with Rhizoctonia

A field trial was conducted in Quitman, Ga. to test the efficacy of theRTI545 addition to a base fungicide/insecticide chemical treatment toprovide Rhizoctonia protection compared to the untreated seed or thisbase or the positive control compromising the synthetic base plussedaxane.

Soybean seeds of the soybean variety cv. Pioneer 93Y92 were treated withseparate slurries being prepared for the chemical and biologicaltreatments which were simultaneously applied to the seed. Seeds wereplaced in a jar which was shaken on a modified paint shaker until theproduct was uniformly coated on the seeds. The base chemical treatmentcomprised four actives comprising 1) 12.8 g/L of fludioxonil, 2) 38.4g/L of mefenoxam, 3) 38.4 g/L of thiophanate-methyl (TPM) and 4) 256 g/Lof thiamethoxam as a single formulation and applied at 41.4 mL/140,000seeds. Vibrance (500 g/L of sedaxane) was applied separately as a seedtreatment to the positive control treatment. Dry technical of the strainRTI545 was used and diluted in water to achieve an application rate of5×10⁵ cfu/seed. Products were slurried with water to at least to 65mL/140,000 seeds to ensure uniform application. The trial comprisedrandomized complete block tests (4 replicates) being 1.8 meters by 9meters plots. Seeds of the soybean variety cv. Treated and untreatedseeds were planted in sandy soil using a cone planter at a seeding rateof 13 seeds/meter at a depth of 2.5 cm and row spacing of 90 cm.Inoculum of Rhizoctonia was mixed and planted with the seed. The plotsand soybeans plants were treated under generally accepted agronomicconditions for soybean including conventional tillage and withsupplemental irrigation as needed. The plants were assessed during thegrowing period for emergence, vigor (on a 1-5 qualitative scale with 5being best) and then harvested to assess yield converted to bu/acre andkg/hectare. The results are summarized in the Table XVII below.

TABLE XVII Impact of RTI545 seed treatment on growth and yield ofsoybeans inoculated with Rhizoctonia Description Emergence Vigor YieldRating Unit % 1-5 BU/Acre Kg/Ha Plant-Eval Interval (DAP) 6 14 18 6 1418 131 Untreated Seed 22 20 16 1.8 1.8 1.3 29.8 2004 Base Synthetic(Base) 71 57 50 2.8 2.3 2.8 48.9 3289 Base + RTI545 5 × 86 60 60 3.8 4.84.5 49.6 3336 10⁵ cfu/seed Base + Vibrance 83 65 62 4.0 4.3 4.3 52.83551

The impact of Rhizoctonia is to reduce stand, reduce vigor and loweryields. Inoculation was effective in view of the base treatment beingsignificantly improved over the untreated seed for all plant counts,vigor and yield assessments.

The addition of RTI545 to the chemical base resulted in significantincrease in stand at all 3 assessment timings, vigor to increasesignificantly at 2 assessment timings and numerically higher yieldcompared to the chemical base. Reduced stand and vigor ratings may bedue to plants dying off. The addition of sedaxane to the synthetic base,an active registered for Rhizoctonia control, resulted in increasedemergence, vigor and numerically higher yield than the base synthetic.RTI545 addition was similar to the sedaxane treatment for all ratingsdemonstrating this strain equal effectiveness in reducing damage fromRhizoctonia in this trial.

Example 13 Impact of Bacillus thuringiensis RTI 545 Seed Treatment onGrowth and Yield of Corn Planted into a Field Infested with Wirewormsand Seed Maggots

A field trial was conducted in Wisconsin to test the efficacy of theRTI545 in addition to either a base fungicide (Maxim and Apron XL bothapplied at 5.2 mL/100 kg) or this base fungicide and clothianidin (0.25mg/seed) or this base fungicide plus bifenthrin (0.125 mg/seed) or thisbase fungicide plus chlorantraniliprole (2 rates evaluated for 0.25 and0.5 mg/seed) or bifenthrin (0.125 mg/seed) combined withchlorantraniliprole (0.25 mg/seed). As a positive control to demonstratea high level of protection, clothianidin was applied at 1.25 mg/seedcombined with the base fungicide with this high rate representing 5-foldthe loading of the low rate of clothianidin also evaluated.

Corn seeds (variety cv. Ag Venture G5891) were treated with separateslurries being prepared for the chemical and biological treatments whichwere simultaneously applied to the seed. Seeds were placed in a jarwhich was shaken on a modified paint shaker until the product wasuniformly coated on the seeds. The base chemical treatment comprisedthree commercial formulations comprising 1) 40.3% of fludioxonil, 2)33.3% of mefenoxam, 3) 18.4% of chlorantraniliprole, 4) 600 g/L ofclothianidin, and 5) 400 g/L of bifenthrin. Dry technical of the strainRTI545 was used and diluted in water to achieve an application rate1×10⁶ cfu/seed. Products were slurried to at least to 600 mL/100 kg toensure uniform application. Treatment 13 is Base+Clothianidin 1.25mg/seed as a positive control for a high level of wireworm protection.The trial comprised randomized complete block tests (4 replicates) of 3meters by 15 meters plots. The corn seeds were planted in Milford siltyclay loam soil using a cone planter at a seeding rate of 93,900seeds/hectare, a depth of 6.25 cm and row spacing of 75 cm. Manure wasspread on top of plots after seeding to attract seed maggots to lay eggsin the plots. The plots and corn plants were treated under generallyaccepted agronomic conditions for corn including minimum tillage withrainfall being average to above average for the area. The plants wereassessed during the growing period for emergence, vigor (on a 1-5qualitative scale with 1=low vigor and 5=high vigor—fullness of plotsrated), feeding damage by wireworms and seed maggot (rated by sampling50 plants and assessing for root damage by wireworm or seed maggot), andthen harvested to assess yield. The results are summarized in TableXVIII.

TABLE XVIII Impact of RTI545 seed treatment on growth and yield of cornplanted into a field infested with wireworms and seed maggot DescriptionWireworm Seed % Damage Maggot Yield Emergence % Stand Vigor % Damage %Bu/A Plant-Evaluation Interval (Days after Planting) 18 28 42 164 19 2842 28 42 28 42 164 1. MAXIM + APRON XL (Base) 49 60 62 57 3.0 3.0 3.017.4 33.6 23.6 22.5 149.3 2. Base + 52 65 69 63 3.3 3.3 3.3 11.2 23.718.7 21.3 170.9 RTI545 1 × 10⁶ cfu/seed 3. Base + 63 77 82 72 3.5 4.04.0 8.6 17 12.4 12.5 210.4 Clothianidin 0.25 mg/seed 4. Treatment 3 + 7691 91 80 4.5 4.5 4.8 5 4.2 4.2 3.8 225.8 RTI545 at 1 × 10⁶ cfu/seed 5.Base + 68 81 82 72 4.0 4.0 4.0 7.3 10.8 8.3 12.5 211.1 Bifenthrin 6.Treatment 5 + 74 88 90 79 4.3 4.3 4.5 7.3 9.7 6.1 7.5 226 RTI545 at 1 ×10⁶ cfu/seed 7. Base + 63 76 78 69 3.5 4.0 4.0 10 16.2 12.4 12.5 198.6Chlorantraniliprole 0.25 mg/seed 8. Treatment 7 + 70 82 83 73 4.0 4.04.0 8.6 12.4 8.3 10 213.4 RTI545 at 1 × 10⁶ cfu/seed 9. Base + 69 82 8272 4.0 4.0 4.0 8.6 10.8 8.3 11.3 209.7 Chlorantraniliprole 0.5 mg/seed10. Treatment 9 + 80 93 95 83 4.8 4.8 5.0 6.1 7.3 3.2 3.8 235.6 RTI545at 1 × 10⁶ cfu/seed 11. Base + 72 84 86 76 4.0 4.0 4.0 10 11.2 7.3 8.8217.3 Chlorantraniliprole 0.25/Bifenthrin 12. Treatment 11 + 82 94 95 835.0 5.0 5.0 5 7.3 3.2 2.5 237.7 RTI545 at 1 × 10⁶ 6 cfu/seed 13. Base +76 91 93 81 4.5 4.5 4.8 5 4.2 6.1 3.8 229.2 Clothianidin 1.25 mg/seed

The impact of wireworm feeding is to reduce stand, reduce vigor andstunt plants by root pruning, damage roots and reduce yields. Seedmaggots can also reduce stand and feed on seed, causing stunting.

The addition of RTI545 to the fungicide base (treatment 2 versustreatment 3) resulted in emergence counts that were significantly higherat 42 and 164 days after planting, reduced the root damage caused bywireworm feeding and significantly increased yield over the fungicidebase. Although RTI545 provided insecticide protection, the level ofprotection was lower than synthetic products evaluated demonstrating thebenefit of adding this product to an insecticide to ensure satisfactoryperformance.

The addition of RTI545 to clothianidin (treatments 3 and 4 respectively)applied at 0.25 mg/seed significantly increased emergence at all 4assessment timings, significantly increased vigor at all 3 assessmenttimings, reduced feeding damage caused by seed maggot and wireworm andsignificantly increased yield. The addition of RTI545 to the low rate ofclothianidin resulted in similar performance to the high rate ofclothianidin applied at 1.25 mg/seed.

The addition of RTI545 to bifenthrin (01.25 mg/seed for treatments 5 and6 respectively) significantly increased emergence at 1 assessmenttimings, significantly increased vigor at 1 assessment timing,significantly reduced feeding damage caused by seed maggot at the lastassessment timing and significantly increased yield.

The addition of RTI545 to chlorantraniliprole (0.25 mg/seed fortreatments 7 and 8 respectively) significantly increased emergence at 3assessment timings and significantly increased yield.

The addition of RTI545 to chlorantraniliprole (0.5 mg/seed fortreatments 9 and 10 respectively) significantly increased emergence atall 4 assessment timings, significantly increased vigor at all 3assessment timings, reduced significantly feeding damage caused by seedmaggot, reduced numerically wireworm feeding damage and significantlyincreased yield. The addition of RTI545 to chlorantraniliprole (0.5mg/seed) resulted in this product performing similarly to the high rateof clothianidin (1.25 mg/seed).

The addition of RTI545 to a chlorantraniliprole and bifenthrincombination (treatments 9 and 10 respectively) significantly increasedemergence at all 4 assessment timings, significantly increased vigor atall 3 assessment timings, reduced significantly feeding damage caused byseed maggot and wireworm and significantly increased yield. The additionof RTI545 this combination resulted in this product to perform similarlyor improved to the high rate of clothianidin (1.25 mg/seed) with thefinal yield of this treatment being over 8 bu/acre higher than the highrate of clothianidin.

These results demonstrate the insecticide protection provided by RTI545enhances protection provided by three unique IRAC classes of syntheticinsecticides. These results demonstrate that RTI545 provides protectionby itself; however, the combination with an effective insecticide helpsensure a much higher level of benefits being apparent.

Example 14 In-Furrow Applications of Bacillus thuringiensis RTI545 inCorn Field Trials

In-furrow treatments of corn with RTI545 were studied in multiple fieldtrials in various locations in North America to evaluate effectivenessagainst wireworm (Melanotus spp.) and corn rootworm (Diabrotica spp.).The effects on yield and crop damage of several treatments compared tountreated controls for the individual North America trials wereaggregated and summarized as an average for the trial in the followingtables as % yield increase and % reduction in damage compared to theuntreated controls. Seven trials were evaluated for wireworm. Eighteentrials were evaluated for corn rootworm. Five trials with heavy damagefrom corn rootworm were averaged separately.

TABLE XIX Effect on corn yield of treatments with RTI545 onwireworm-infested field plots % Yield Treatment Rate Increase Bifenthrin56 g ai/Ha 5 RTI545 3 × 10¹¹ CFU/Ha 1 3 × 10¹² CFU/Ha 2 Bifenthrin + 56g ai/Ha + 3 × 10¹¹ CFU/Ha 6 RTI545 56 g ai/Ha + 3 × 10¹² CFU/Ha 7Tefluthrin 168 g ai/Ha 5

TABLE XX Effect on corn yield and rootworm damage of treatments withRTI545 on corn rootworm-infested field plots Heavy CRW Pressure % Yield% Damage % Yield % Damage Rate Increase reduction Increase reductionBifenthrin 112 g ai/Ha 4 42 11 45 RTI545 1.24 × 10¹² CFU/Ha 3 28 11 356.18 × 10¹² CFU/Ha 4 37 11 44 Bifenthrin + 112 g ai/Ha + 4 49  9 51RTI545 1.24 × 10¹² CFU/Ha 112 g ai/Ha + 6 55 22 62 6.18 × 10¹² CFU/HaTefluthrin 168 g ai/Ha 3 46 16 52

Yield data from 7 wireworm trials indicate treatments containing RTI545show a slight advantage over bifenthrin alone and commercial standardtefluthrin at its highest rate. Data from 18 trials for corn rootwormindicate yields from treatments containing RTI545 show yields equal toor slightly higher than bifenthrin alone, and tefluthrin at its highestrate. Data from these 18 trials also indicate that corn treated withRTI545 in combination with an insecticide resulted in less root damageto corn in corn rootworm infested soil than the industry standard oftefluthrin at its maximum rate or bifenthrin at maximum rate or whenRTI545 was applied alone as a treatment. When there was heavy cornrootworm pressure, the reduction in damage ratings was similar to thetreatments under typical rootworm pressure, but the percentage yieldincrease was better. Overall, the data from these trials indicate thatRTI545 combined with bifenthrin can enhance corn yield and decreaseinsect damage to corn roots when compared to untreated areas or areastreated with the industry standard tefluthrin.

Field trials were also conducted with in-furrow treatments of corn withRTI545 in Europe to evaluate effectiveness against wireworm (Agriotesspp.)—Data not shown.

Example 15 Impact of Bacillus thuringiensis RTI545 on Growth and Yieldof Peanut Inoculated with Rhizoctonia

A field trial was conducted in Georgia to test the efficacy of thebiological combination of the invention as a seed treatment of peanutinoculated with Rhizoctonia solani.

Peanut seeds were treated with a dry dust formulation with a finalapplication rate of 200 g/100 kg of seed containing RTI545, resulting inan application of RTI545 at 3.0×10⁶ CFU/g of seed, and simultaneousapplied with DYNASTY PD, a United States registered peanut productcontaining fludioxonil (2%), mefenoxam (0.4%) and azoxystrobin (3.2%)with an application of 195 g/100 kg of seed. Seeds were placed in a jarand shaken until the product was uniformly coated on the seeds.

The trial comprised randomized complete block tests using 6 foot by 30foot (about 1.8 meters by 9.1 meters) plots. Treated and untreatedpeanut (Arachis hypogaea, var. GA 06) seeds were planted. Using a coneplanter, seeds were planted 1.25 inch deep (3 cm) with row spacing of 36inches (0.9 m) in sandy loam soil. The plots were inoculated withRhizoctonia sp. to ensure infestation. The plots and peanut plants weretreated under generally accepted agronomic conditions for peanutincluding conventional tillage and irrigation as needed until they wereready for harvest. The plants were assessed during the growing periodfor emergence, vigor (on a 1-5 qualitative scale), and then harvested toassess yield. The results are summarized in Table XXI.

TABLE XXI Results from field trial of RTI545 seed treated peanutsagainst Rhizoctonia Base Timing Untreated Base Chemical + Rating DAPSeed Chemical RTI545 Plant Counts   8 3 10 12  11 11 27 39  16 15 41 45Vigor (1-5)   8 1.8 3.0 3.3  11 2.3 2.8 4.0  16 1.8 2.5 3.5 Yieldlb/Acre 135 1277 3231 3733 Kg/Hectare 1434 3628 4191

All seed treatments had a higher stand than the untreated control (UTC)at all 3 assessment timings. At all three assessment timings, thetreatment including RTI545 provided the best emergence. The untreatedseed had the lowest vigor at all 3 assessment timings. The treatmentincluding RTI545 provided stronger vigor than the UTC and the basechemical treatment. All products provided significantly higher yieldthan the UTC. The treatment including RTI545 outperformed base chemicaltreatment.

Example 16 Impact of Bacillus thuringiensis RTI 545 Seed Treatment onLesion Nematode

Soil assays on seed-treated corn to assess activity against lesionnematode (Pratylenchus penetrans) were conducted in a greenhouse. Corn(cv. Viking) seeds were treated with a base seed treatment offludioxonil (1.1 ml/SU) and metalaxyl-M (1.1 ml/SU) (all seeds). Someseeds were also treated with PONCHO/VOTIVO (clothianidin+B. firmus1-1582 at 80 ml/SU), RTI545 at 5×10⁵ CFU/seed, RTI545 at 1×10⁶ CFU/seedor a mixture of either RTI545 (5×10⁶ CFU/seed)+Bacillus subtilis strainCH201 (2.5×10⁶ CFU/seed)+Bacillus licheniformis strain CH200 (2.5×10⁶CFU/seed) or RTI545 (5×10⁶ CFU/seed)+Bacillus velezensis strain RTI301(5×10⁵ CFU/seed)+Bacillus licheniformis strain CH200 (2.5×10⁶ CFU/seed).

In one type of assay, the treated seeds were planted in cone-shapedcontainers in 90 ml of soil (80.4% sand, 14.8% silt, 4.8% clay, organicmatter 1.1%, pH 6.9). After planting the seeds, the soil was inoculatedon the same day with 4000 nematodes (mixed stages: J2-adults) per seedin 200 μl of carrier (2% methyl cellulose). A control used seeds withthe base seed treatment in non-inoculated soil. The test plantings weretop watered using a mist sprinkler. The test was evaluated at 2 (datanot shown) and 7 days after emergence for total root length usingWinRhizo™ software. Root length data and nematode % reduction data weretransformed using the arcsine square root transformation prior to ANOVAanalysis using an ANOVA Fisher test at α=0.1.

TABLE XXII The effect of RTI545 seed treatment in corn on the total rootlength of seedlings grown under lesion nematode pressure. % Root lengthIncrease vs Treatment inoculated control Base seed treatment, inoculated 0 Base seed treatment, non-inoculated 18 Base seed treatment +PONCHO/VOTIVO 19 Base seed treatment + RTI545 (5 × 10⁵ CFU/seed) 30 Baseseed treatment + RTI545 (1 × 10⁶ CFU/seed) 34

Inoculation of corn seedlings with lesion nematodes numerically reducedtotal root length. The total root length of seedlings treated withPONCHO/VOTIVO (commercial standard) was greater than the non-treatedinoculated seedlings and similar to non-treated non-inoculatedseedlings. The total root length of RTI545-treated seedlings wassignificantly increased when compared with non-treated inoculatedseedling (30-34%) and was statistically similar but numerically greaterthan the total root length of seedlings treated with PONCHO/VOTIVO.

Similar tests using 2000 nematodes/seed for inoculation were carried outin 130 ml of soil and measured nematode numbers per root and fresh topweight of the plants at 8 weeks after nematode inoculation. The resultsare presented in Table XXIII. Lesion nematode infestation resulted insevere reduction of the fresh top weight (FTW) of corn plants. The FTWof inoculated control plants was reduced by 50% when compared withnon-inoculated plants. The high rate of RTI545 provided statisticallygreater FTW compared to standards PONCHO/VOTIVO and to inoculatednematode control. These results indicate that RTI545 seed treatmentincreases plant tolerance to nematode infestation by sustaining plantgrowth in the presence of nematode pests. In addition, similar positiveeffects on plant growth/FTW were observed where RTI545 was used at thelower rate in combination with other strains: RTI545 (1×10⁶CFU/seed)+Bacillus subtilis strain CH201 (2.5×10⁶ CFU/seed)+Bacilluslicheniformis strain CH200 (2.5×10⁶ CFU/seed), or RTI545 (1×10⁵CFU/seed)+RTI301 (5×10⁵ CFU/seed)+Bacillus licheniformis strain CH200(2.5×10⁶ CFU/seed). Moreover, RTI545 seed treatment at a high rate(1.0×10⁶ CFU/seed) provided very good control of lesion nematodes (50%reduction of nematode numbers in the roots). PONCHO/VOTIVO did notreduce nematode numbers in roots.

TABLE XXIII The effect of RTI545 seed treatment in corn on fresh topweight and nematode counts in roots after inoculation with lesionnematode. % Fresh Reduction Top in Weight Treatment Penetration (g) Baseseed treatment, inoculated —  6.5 Base seed treatment, non-inoculated  012.8 Base seed treatment + PONCHO/ No control  8.5 VOTIVO Base seedtreatment + RTI545  8 11.6 (5 × 10⁵ CFU/seed) Base seed treatment +RTI545 49 13.3 (1 × 10⁶ CFU/seed) Base seed treatment + RTI545 12 13.2(5 × 10⁵ CFU/seed) + CH200(2.5 × 10⁶ CFU/seed) + CH201 (2.5 × 10⁶CFU/seed) Base seed treatment + RTI545  6 12.1 (5 × 10⁵ CFU/seed) +RTI301 (5 × 10⁵ CFU/seed) + CH200 (2.5 × 10⁶ CFU/seed)

Similar tests wherein RTI545 was applied as a soil drench at the rate of2.5×10¹¹ CFU/ha provided 71% reduction in lesion nematode numbers perroot compared to 95% for abamectin. In soil drench tests applied to potsin the greenhouse with soil infested with lesion nematode eggs andadults (2000 individuals/pot) RTI545 at the rate of 2.5×10¹³ CFU/haprovided 80% reduction in nematode numbers per root compared to 86%reduction by cadusafos.

Example 17 Impact of Bacillus thuringiensis RTI 545 on Soybean CystNematode in Soil Drench Assays

The activity of RTI545 against soybean cyst nematode (Heteroderaglycines) was investigated in soil drench assays in a greenhouse.Individual soybean (cv. AG4730) seeds were planted in 120 ml soil (80.4%sand, 14.8% silt, 4.8% clay, organic matter 1.1, pH 6.9) in cone-shapedcontainers and watered with individual bottom watering. The containerswere inoculated with nematode eggs at a rate of 4000 eggs in 1 mL of 2%methyl cellulose per container 21 days after planting. Soil drench (in10 ml volume per 100 ml soil) applications of RTI545 and AGRI-MEK 0.15EC (a.i. 2% abamectin) and VENERATE XC (94.46% heat-killed Burkholderiaspp. strain A396 cells and spent fermentation media) were applied at 7and 21 days after planting. The rates tested were RTI545 washed sporesat 2.5×10¹² CFU/ha, 2.5×10¹³ CFU/ha and 2.5×10¹⁴ CFU/ha; abamectin at 1ppm (0.01 mg a.i./plant) and 10 ppm (0.1 mg a.i./plant) corresponding toseed treatment label rate; and VENERATE XC at 5% v/v (500 mg/plant),corresponding to 4.5× the in-furrow rate. The tests were evaluated atday 70 (7 weeks after inoculation). Evaluations were carried out byextraction of cysts from roots and soil and counting the total number ofcysts under a stereomicroscope.

TABLE XXIV Number of cysts of soybean cyst nematode extracted from rootsand soil. Number of cysts % Reduction Treatment extracted of cystsUntreated, non-inoculated   0 — Untreated, inoculated 147 na RTI545 (2.5× 10¹² CFU/ha)  50 66 RTI545 (2.5 × 10¹³ CFU/ha)  93 37 RTI545 (2.5 ×10¹⁴ CFU/ha)  66 55 Abamectin (0.01 ppm)  31 79 Abamectin (0.1 ppm)   796 VENERATE XC (500 mg/plant)  43 71

The data show that RTI545 reduced nematode cyst numbers up to 66%. Therewas no clear dose response between the rates tested and activity. Theactivity of RTI545 was not statistically different from the biologicalstandard VENERATE XC. Chemical standard abamectin had the highestactivity. The rate of 0.01 mg/plant provided 79% reduction and 0.1mg/plant provided 96% reduction.

Example 18 Suspension Concentrate Formulations Comprising Bacillusthuringiensis RTI545

Representative suspension concentrates comprising Bacillus thuringiensisRTI545 are summarized in Table XXV. They were prepared by mixing sporesof RTI545 with the other components in a suitable mixing vessel orhomogenizer.

TABLE XXV Suspension Concentrate Formulations Example 18A 18B ComponentFunction % (w/w) RTI545 3 × 10¹¹ cfu/g Active ingredient  5.25 9.59Ammonium sulfate Antifreeze 9.5 Glycerol (86.5%) Antifreeze 48Attapulgite (20-35% aq. Thickener 9.0 suspension) Alkyl polyglycosides,Dispersant 8.0 mixture Anionic Phosphate ester Dispersant 7.0surfactants, mixture Aq. Dispersion of ethylene Dispersant 0.89 vinylacetate copolymer silicone emulsion antifoam 0.3 Potassium sorbatePreservative 0.1 0.2 Water Diluent 59.54 38.82

Example 19 Suspension Concentrate Formulations Comprising Bacillusthuringiensis RTI545 and Bifenthrin

Representative suspension concentrates comprising Bacillus thuringiensisRTI545 and bifenthrin insecticide are summarized in Tables XXVI andXXVII. They were prepared by mixing spores of RTI545 with the othercomponents in a suitable mixing vessel or homogenizer. Example 19C is afoamable composition that can be applied as a foam to seeds or in-furrowat time of planting. The foamable composition 19C can be optionallydiluted with water and mixed with a pressurized gas such as air in afoaming chamber comprising a foaming medium such as a plurality of glassbeads to prepare a foam.

TABLE XXVI SC formulations of RTI545 and bifenthin Example 19A 19B 19CComponent Function % (w/w) Technical B fenthr n Active ingredient (99%)15.81 Active ingredient (98.2%) 15.92 15.96 RTI545 3 × 10¹¹ cfu/g Activeingredient 3.62 5.25 5.0 Ammonium sulfate Antifreeze 10.75 9.5 GlycerinAntifreeze 12.7 Thickener 2.15 Attapulgite Thickener, 20-35% aq. 9.0suspension Xanthan gum Thickener, 2% suspension 12 Alkyl polyglycosides,Dispersing agent 6.00 8.0 1.25 mixture Anionic Phosphate esterDispersing agent 1.57 7.0 1.25 surfactants, mixture Sodium decylsulphate Foaming agent 20 35-40% in water silicone emulsion Antifoam 0.10.3 1,2-benzisothiazolin-3-one Preservative, 20% alkaline 0.15 solutionPreservative, 20% aqueous 0.1 solution Kathon ® CG-ICP Preservative 0.1sodium salt o-phenylphenate Preservative 0.1 Potassium sorbatePreservative 0.1 Sodium benzoate Preservative 0.1 Acetic acid Diluent 01.21 0 Water Diluent 59.85 43.62 31.54

TABLE XXVII Example 19D 19E Component Function % (w/w) TechnicalBifenthrin Active ingredient (99%) 17.4 16.6 RTI545 3 × 10¹¹ cfu/gActive ingredient  5.0 10 Ammonium sulfate Antifreeze 11.2 10.6 Alkylpolyglycosides, mixture Dispersing agent 12.9 12.3 Anionic Phosphateester Dispersing agent  1.3 1.2 surfactants, mixture silicone emulsionAntifoam  0.3 0.3 Potassium sorbate Preservative  0.1 0.1 Sodiumbenzoate Preservative  0.1 0.1 Citric acid Diluent  0.8 0.7 H₃PO₄Diluent  0.4 0.5 Water Diluent 50.4 47.5

Spore stability during storage at elevated temperatures was very good,as shown below in Table XXVIII, in which formulation 18D was stored at54° C. for two weeks, with little change in the concentration of RTI545Ssores.

TABLE XXVIII Spore stability during storage Spore stability Initial 2weeks storage at 54° C. 1.36 × 10¹⁰ cfu 1.30 × 10¹⁰ cfu

All publications, patent applications, patents, and other referencesmentioned in the specification are indicative of the level of thoseskilled in the art to which the presently disclosed subject matterpertains. All publications, patent applications, patents, and otherreferences are herein incorporated by reference to the same extent as ifeach individual publication, patent application, patent, and otherreference was specifically and individually indicated to be incorporatedby reference. It will be understood that, although a number of patentapplications, patents, and other references are referred to herein, suchreference does not constitute an admission that any of these documentsforms part of the common general knowledge in the art.

Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

That which is claimed:
 1. A composition comprising a biologically pureculture of Bacillus thuringiensis RTI545 deposited as ATCC No.PTA-122161, and an agriculturally acceptable adjuvant, which improvesthe stability of the Bacillus thuringiensis RTI545; for application to aplant for one or both of benefiting plant growth or conferringprotection against a plant pest in a susceptible plant.
 2. Thecomposition of claim 1, wherein the biologically pure culture ofBacillus thuringiensis RTI545 is in the form of spores or vegetativecells.
 3. The composition of claim 1, wherein the adjuvant comprises aplanting matrix, a carrier, a binder, a surfactant, a dispersant, or ayeast extract.
 4. The composition of claim 1, further comprising aninsecticide, a fungicide, nematicide, bacteriocide, biostimulant,herbicide, plant extract, microbial extract, plant growth regulator,fertilizer or combinations thereof present in an amount suitable tobenefit plant growth and/or to confer protection against a pathogenicinfection in a susceptible plant.
 5. The composition of claim 1, whereinthe composition further comprises a chemical insecticide.
 6. Thecomposition of claim 5 wherein the chemical insecticide compriseschlorantraniliprole, chlorethoxyfos, chlorpyrifos-e, cyantraniliprole,cyclaniliprole, cypermethrin, dichloropropene, flupyradifurone,gamma-cyhalothrin, profenofos, tebupirimfos, tefluthrin,kappa-bifenthrin, kappa-tefluthrin, carbofuran, carbosulfan, oxamyl,thiodicarb, chlorpyrifos, chlorpyrifos-e, chlorpyrifos-methyl, diazinon,phorate, terbufos, fipronil, acetamiprid, clothianidin, imidacloprid,thiacloprid, thiamethoxam, abamectin, flonicamid, flubendiamide,bifenthrin, lambda-cyhalothrin, cypermethrin, zeta-cypermethrin,deltamethrin, pyridaben or any mixtures thereof.
 7. The composition ofclaim 6, wherein the chemical insecticide comprises bifenthrin.
 8. Thecomposition of claim 1, wherein the composition further comprises achemical fungicide.
 9. The composition of claim 8 wherein the chemicalfungicide comprises thiabendazole, fluxapyroxad, penflufen, sedaxane,bitertanol, cyproconazole, difenoconazole, fluquinconazole, flutriafol,ipconazole, myclobutanil, prothioconazole, triadimefon, triadimenol,tebuconazole, triticonazole, prochloraz, imazalil, benomyl,carbencladzim, hymexazole, azoxystrobin, fluoxastrobin, pyraclostrobin,trifloxystrobin, carboxin, flutolanil, metalaxyl, mefenoxam,penthiopyrad, fluopyram, silthiofam, fluazinam, pyrimethanil,fludioxonil, iprodione, tricyclazole, captan, dammet, mancozeb, metam,thiram, guazatine, tolclofos-methyl, pencycuron, thiophanate-methyl,fenpicoxamide, mefentrifluconazole, fluindapyr, or any mixtures thereof.10. The composition of claim 1, wherein the composition furthercomprises a chemical nematicide.
 11. The composition of claim 10,wherein the chemical nematicide comprises benomyl, fenamiphos,cadusafos, ethoprophos, fosthiazate, chloropicrin, dammet, fluensulfone,oxamyl, 1,3-dichloropropene (telone), metam sodium, metam potassium,metam salt, methyl bromide, allyl isothiocyanate, fluazaindolizine,tioxazafen, fluopyram, or any mixtures thereof.
 12. The composition ofclaim 1, wherein the composition is in a formulation compatible with aliquid fertilizer or crop nutrition product.
 13. The composition ofclaim 12, wherein the composition further comprises a hydratedaluminum-magnesium silicate and at least one dispersant.
 14. Thecomposition of claim 12 or 13, comprising a bifenthrin insecticidewherein the bifenthrin is present at a concentration ranging from 0.1g/ml to 0.2 g/ml.
 15. A method for one or both of benefiting growth of aplant or conferring protection against a plant pest in a susceptibleplant, the method comprising delivering a composition comprising abiologically pure culture of Bacillus thuringiensis RTI545 deposited asATCC No. PTA-122161, to the plant, plant part, seed of the plant, rootsof the plant, soil or growth medium surrounding the plant, or soil orgrowth medium before planting the plant or sowing seed of the plant, inan amount suitable to benefit the plant growth and/or to conferprotection against the plant pest in the susceptible plant.
 16. A methodfor one or both of benefiting growth of a plant or conferring protectionagainst a plant pest in a susceptible plant, the method comprising:planting a seed of the plant, wherein the seed has been coated with acomposition comprising a biologically pure culture of Bacillusthuringiensis RTI545 deposited as ATCC PTA-122161, wherein growth of theplant from the seed is benefited and/or protected against the plant pestis conferred.
 17. The method of claim 15 or 16, wherein the plantcomprises corn, soybean, potato, cotton, tomato, pepper, cucurbit,sugarcane, peanut or wheat.
 18. The method of claim 15 or 16, whereinthe plant pest comprises an insect selected from the group consisting ofLygus spp., Coleoptera, Diabrotica spp., Melanotus spp., Phyllophagaspp., Limonius spp., Agriotes spp., Lepidoptera, Peridroma spp., Euxoaspp., Agrotis spp., Diptera, Hylemya spp., Tetanops sp. Hemiptera,Pemphigus sp., Aphis sp., Agonoderus sp., Feltia spp., or combinationsthereof.
 19. The method of claim 18, wherein the plant pest comprises aplant bug, rootworm, wireworm, soil-dwelling maggot, or white grubcomplex.
 20. The method of claim 15 or 16, wherein the plant pestcomprises a plant pathogenic nematode selected from the group consistingof Rotlyenchulus spp., Xiphinema spp., Hoplolaimus spp., Paratylenchusspp., Criconemoides spp., Meloidogyne spp., Hemicycliophora spp.,Helicotylenchus spp., Trichodorus spp., Heterodera spp., Belonolaimusspp., Tylenchorhynchus spp., Globodera spp. or combinations thereof. 21.The method of claim 20 wherein the nematode comprises a Meloidogyne sppnematode, Pratylenchus spp. nematode, or Globodera spp. nematode orHeterodera spp. nematode.
 22. The method of claim 15 or 16, wherein theplant pest comprises a plant fungal pathogen or a plant bacterialpathogen selected from the group consisting of Alternaria ssp.,Aspergillus spp., Botrytis spp, Cercospora spp., Fusarium spp.,Phytophthora spp., Rhizoctonia spp., Magnaporthe spp., Pythium spp.,Monilinia spp., Colletotrichum spp., Sclerotinia spp., and Erwinia spp.23. The method of claim 22 wherein the plant fungal pathogen comprisesRhizoctonia spp.
 24. The method of claim 15 or 16, further comprisingdelivering a liquid fertilizer or crop nutrition product to the seed ofthe plant.
 25. A plant seed coated with a composition comprising sporesof a biologically pure culture of Bacillus thuringiensis RTI545deposited as ATCC No. PTA-122161, present in an amount suitable tobenefit plant growth and/or to confer protection against a plant pest ina susceptible plant.
 26. The plant seed of claim 25, wherein the seedcomprises the seed of corn, soybean, potato, cotton, tomato, pepper,cucurbit, sugarcane, peanut or wheat.